Jan Knol

Karin Bodewits

Gerda I Hessels

Lubbert Dijkhuizen
email: l.dijkhuizen@rug.nl

Robert Van

Robert Van Der Geize

Keto-5α-steroid ∆1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism

Keywords: 3-ketosteroid dehydrogenases, 5α-androstane-3, 17-dione, cholesterol degradation, steroids, actinomycetes, Rhodococcus

Introduction

3-Ketosteroid ∆ 1 -dehydrogenase (KSTD) [4-ene-3-oxosteroid: (acceptor)-1-eneoxoreductase, EC 1.3.99.4] is a flavoprotein catalyzing the trans-axial elimination of the C1(α) and C2(β) hydrogen atom of the A-ring from the poly-cyclic ring structure of 3ketosteroids [START_REF] Itagaki | Spectral properties of 3-ketosteroid ∆ 1 -dehydrogenase from Nocardia corallina[END_REF]. KSTD is a key enzyme in microbial steroid catabolism needed for opening of the steroid B-ring [2][START_REF] Hörhold | Über den Abbau von Steroiden durch Mikroorganismen[END_REF][START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF][START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]. KSTD activities have been found in various actinobacteria, e.g. Arthrobacter [START_REF] Mólnar | Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-∆ 1 -dehydrogenase, 3-ketosteroid-∆ 5 -isomerase and a hypothetical regulatory protein[END_REF], Mycobacterium [7] and Rhodococcus [START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF][START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF][START_REF] Morri | 3-ketosteroid-∆ 1 -dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme[END_REF][START_REF] Van Der Geize | Unmarked gene deletion mutagenesis of kstD, encoding 3ketosteroid ∆1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker[END_REF][START_REF] Mcleod | The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse[END_REF], as well as in Comamonas (formerly Pseudomonas) testosteroni [START_REF] Plesiat | Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid ∆1-dehydrogenase[END_REF][START_REF] Horinouchi | A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroid and 3ketosteroid dehydrogenase genes[END_REF]. Choi et al. (1995) [13] proposed to classify KSTD enzymes into two groups, based on their ability to use 11-substituted steroids as substrate. KSTD of P. testosteroni is unable to desaturate C11hydroxy-or 11-ketosteroids [START_REF] Levy | Bacterial oxidation of steroids I: ring A dehydrogenations by intact cells[END_REF], while KSTD of Nocardia corallina acts on C11substituted steroids [START_REF] Itagaki | Purification and characteriztion of 3-ketosteroid-∆ 1 -dehydrogenase from Nocardia corallina[END_REF]. Cortisone (11-keto), but not 11β-hydrocortisone, was found to be an excellent substrate. Characterized KSTD enzymes display quite a broad substrate range [7,13,[START_REF] Itagaki | Purification and characteriztion of 3-ketosteroid-∆ 1 -dehydrogenase from Nocardia corallina[END_REF][START_REF] Sih | Steroid 1-dehydrogenase of Nocardia restrictus[END_REF]. Highest ∆1 KSTD activities are observed with steroid substrates carrying the 3-keto-4-ene structure; (9-hydroxy-)4-androstene-3,17-dione (4-AD) and 17β-hydroxy-4-androstene-3-one (testosterone) usually are preferred substrates. ∆1 KSTD activity on saturated 3-ketosteroids, e.g. 5α-androstane-3,17-dione (5α-AD), has been observed in P. testosteroni [START_REF] Florin | Comamonas testosteroni 3-ketosteroid-∆ 4 -dehydrogenase: gene and protein characterization[END_REF]. However, no kinetic data for ∆1 KSTD enzymes and these saturated 3-ketosteroids is available.

In previous work, we have characterized the R. erythropolis SQ1 ∆1 KSTD1 and ∆1 KSTD2 iso-enzymes [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF][START_REF] Van Der Geize | Unmarked gene deletion mutagenesis of kstD, encoding 3ketosteroid ∆1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker[END_REF]. Analysis of a kstD1 kstD2 double gene deletion mutant R. erythropolis strain RG8 showed stoichiometric accumulation of 9α-hydroxy-4-4 androstene-3,17-dione (9OHAD) from 4-AD due to blocked opening of the steroid Aring [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]. 4-AD is one of the proposed central pathway intermediates in the degradation of cholesterol. However, strain RG8 is still able to degrade and grow on cholesterol as sole carbon and energy source without accumulation of steroid pathway intermediates (e.g. 4-AD, 9OHAD) (this study). Our subsequent studies resulted in identification of a third ∆1 KSTD (KSTD3) enzyme in strain RG8.

Analysis of the genome of the human pathogen Mycobacterium tuberculosis H37Rv revealed the presence of a single gene (rv3537) encoding a putative ∆1 KSTD [START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF][START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF][START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF].

M. tuberculosis is the causative agent of tuberculosis causing millions of deaths every year. The rv3537 gene is one of several essential pathogenicity genes recently identified as part of a gene cluster encoding cholesterol catabolism [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF]20]. Biochemical characterization and phylogenetic analysis indicates that Rv3537 of M. tuberculosis H37Rv is the orthologue of ∆1 KSTD3 of R. erythropolis SQ1 (this study). Phylogenetic analysis revealed clustering of ∆1 KSTD enzymes in at least four different groups. Here we report the biochemical characterization of a third and unique ∆1 KSTD (KSTD3) of R. erythropolis SQ1 (and strain RG8) and its orthologue Rv3537 in Mycobacterium tuberculosis H37Rv involved in cholesterol catabolism.

Experimental

Bacterial strains and growth conditions

Rhodococcus strains were cultivated in complex medium at 30ºC and 200 rpm. Complex medium (LBP) contained 1% (wt/vol) bacto-peptone (Difco, Detroit, Mich., USA), 0.5% (wt/vol) yeast extract (BBL Becton Dickinson and Company, Cockeysville, Md.) and 1% (wt/vol) NaCl. Escherichia coli strains were grown in Luria-Bertani (LB, Sigma.

Zwijndrecht, The Netherlands) broth at 37ºC unless stated otherwise.

General cloning techniques

Recombinant DNA techniques were done according to standard protocols [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. E. coli DH5α [START_REF] Hanahan | Studies on transformation of Escherichia coli with plasmids[END_REF] was used as general host for cloning. Transformation of E. coli strains was done as described by [START_REF] Chung | One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same soulution[END_REF] [START_REF] Chung | One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same soulution[END_REF]. DNA modifying enzymes (restriction enzymes, ligase, polymerase) were purchased from Roche (Mannheim, Germany) or New England Biolabs (Beverly, Mass.) and were used as described by the manufacturer.

Isolation of DNA restriction fragments from agarose gels was done using the Qiagen (Basel, Switzerland) gel extraction kit according to protocol. DNA nucleotide sequencing was done by AGOWA (Berlin, Germany). These sequence data have been submitted to the DDBJ/EMBL/GenBank database under accession number EU014895.

Preparation of Rhodococcus cell extracts and KSTD activity staining on native PAGE

Overnight cultures (250 ml) of wild-type R. erythropolis SQ1 [START_REF] Quan | Nocardioform arsenic resistance plasmid characterization and improved Rhodococcus cloning vectors[END_REF] and kstD1 kstD2 double gene deletion mutant R. erythropolis RG8 [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF] were grown in LBP medium until 6 OD 660 of 4 and subsequently induced with steroids (1 g•L -1 ) for an additional 4 h. Cell pellets (30 min; 2500 g; 4˚C) were washed with 200 ml phosphate buffer (2.72 g•L -1 KH 2 PO 4 , 3.48 g•L -1 K 2 HPO 4 , 2.46 g•L -1 MgSO 4 •7H 2 O, pH 7.2). The washed cell suspensions were concentrated to 4 ml and disrupted by passage through a French pressure cell (140 Mpa) twice. Cell extracts were centrifuged for 30 min at 25000 g to remove cell debris. The resulting cell free extracts were used for analysis of KSTD activity on native PAGE (10% acrylamide). KSTD activity (approx. 30 -50 µg protein loaded on gel) was visualized by incubating native PAGE gels in 100 ml 66.7 mM TRIS buffer containing 3.1 mg phenazine methosulphate, 2.9 mg steroid (dissolved in ethanol) and 41 mg nitrobluetetrazolium (NBT, dissolved in 70% dimethylformamide).

Screening a R. erythropolis genomic library for 3-ketosteroid dehydrogenase genes

Degenerate kstD deg PCR primers [forward 5'gtggtcctggcagc(a/c)gg(g/c)gg(g/c)tt and reverse 5'ttgccggcggcgtacaggccnt(c/t)gat] were developed based on conserved sequences of amino acid sequences of ∆1 KSTD proteins and the known nucleotide sequences of kstD1 and kstD2, and used to screen chromosomal DNA of R. erythropolis RG8 for the presence of novel 3-ketosteroid dehydrogenase genes. Specific kstD new primers [forward 5'cttcgtgtccaatcgcgagatg and reverse 5'gacggtggcttgaaggccgg] were designed on the obtained 882 bp PCR fragment from RG8 and used to screen a genomic library of R. erythropolis strain RG1 in plasmid pRESQ [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF] to get a clone containing the full length kstD gene sequence. The genomic library of strain RG1 was introduced into E. coli DH5α and plated on LB with 25 µg/ml kanamycin. All colonies on a plate were used for plasmid isolation [START_REF] Birnboim | A rapid alkaline extraction method for the isolation of plasmid DNA[END_REF] and checked with PCR for positive signal with kstD new primers.

S t a g e 2 ( a ) P O S T -P R I N T

7 PCR was performed using 30 cycles of 1 min 94ºC, 1 min 60 ºC and 1 min 72ºC. This procedure of transformation, plating dilutions and PCR was repeated several times to identify a single clone, containing the pRESQ vector with a 7.4 kb insert containing kstD3, designated pJK2 (Fig. 2).

Heterologous expression of kstD3 in E. coli cells

Total DNA of R. erythropolis was isolated as described [START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF]. PCR on total DNA was performed using 30 cycles of 1 min 94ºC, 1 min 60 ºC and 1. The pET15b-KSTD3 construct was introduced into E. coli strain BL21(DE3) (Invitrogen) resulting in recombinant strain BL21pET15bKSTD3. The kstD1 (see for PCR [START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF]) and kstD2 [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF] genes were also cloned into the NdeI/BamHI site of pET15b and transformed into E.coli BL21(DE3) resulting in recombinant strains BL21pET15bKSTD1 and BL21pET15bKSTD2.

Hundred-fold diluted pre-cultures of recombinant E. coli strains were grown for 48 h at

Results and discussion

Identification of additional KSTD activities in kstD1 kstD2 mutant RG8

Deletion of the 3-ketosteroid-∆1-dehydrogenase genes kstD1 and kstD2 in R. erythropolis SQ1 did not result in accumulation of steroid intermediates during incubation of strain RG8 with cholesterol [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]. We now also observed that strain RG8 still grows normally on cholesterol as sole carbon source. Therefore, we predicted the presence of additional KSTD enzymes in R. erythropolis SQ1. KSTD activity staining, first with 4-AD (a ∆1

KSTD substrate) and subsequently with 1-(5α)-androstene-3,17-dione (1-5α-AD) (a ∆4 KSTD substrate) visualized activity bands for two ∆1 KSTD enzymes and for one ∆4

KSTD enzyme in wild-type R. erythropolis SQ1 (Fig. 1). Only KSTD1 enzyme activity band is missing in the kstD1 kstD2 mutant strain R. erythropolis RG8 (Fig. 1) (see also [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]). The ∆4 KSTD and second ∆1 KSTD enzyme have not been characterized yet. The steroids progesterone (P), 4-AD and 9OHAD each induce two ∆1 KSTD enzymes and one ∆4 KSTD enzyme, although these inducing compounds are substrates for ∆1 KSTD enzymes.

Figure 1 thus demonstrates the natural diversity of 3-ketosteroid-dehydrogenases in R.

erythropolis. The number of KSTD activities shown in Fig. 1 is even an underestimation, since some KSTD enzymes (for instance KSTD2) can not use NBT as electron acceptor and are not detected in this assay [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF].

The two KSTD activities observed in kstD1 kstD2 mutant strain RG8 (Fig. 1) indicate that, in addition to KSTD1 and KSTD2, R. erythropolis harbors at least one more ∆1

KSTD and at least one ∆4 KSTD activity. In subsequent work we have cloned and ), a set of degenerate PCR primers was designed and used for screening strain RG8 for the presence of additional kstD sequences. A PCR product of approximately 0.9 kb was obtained and sequenced. The deduced amino acid sequence was similar, but not identical, to KSTD1 and KSTD2 from R. erythropolis SQ1, suggesting that a third kstD gene had been cloned. The full length gene was subsequently isolated by screening a genomic library of R. erythropolis RG1 [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF] with specific PCR primers based on the nucleotide sequence of the 0.9 kb PCR product. A positive clone (pJK2), carrying a 7.4

kb insert (GC-content 63.3%), was identified and sequenced. Bioinformatic analysis of the nucleotide sequence revealed a total of 6 ORFs, two of which showed similarity to kstD genes (Fig. 2). One ORF (kst4D) encodes a protein displaying high similarity (45% identity) with 3-ketosteroid-∆4(5α)-dehydrogenase of Comamonas testosteroni TA441 [START_REF] Horinouchi | A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroid and 3ketosteroid dehydrogenase genes[END_REF], while the second, divergently transcribed ORF (1,725 nt, GC content 63.6%, kstD3) encodes a protein with high similarity to ∆1 KSTD1 (37% identity) and KSTD2 (42% identity) of R. erythropolis SQ1 (Fig. 2; Table 1; [START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF], [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]). KSTD3 displays highest similarity (78% identity) with Ro04532 of Rhodococcus sp. RHA1. Transcriptomic S t a g e 2 ( a ) P O S T -P R I N T 14 analysis recently showed that ro04532 is upregulated during growth on cholesterol, thus probably catalyzing a ∆1-dehydrogenation reaction during cholesterol degradation [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF].

Four additional ORFs were identified on the cloned DNA fragment, and all appear to be involved in cholesterol catabolism (Fig. 2; Table 1; [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF]). The choG gene encodes a protein highly similar (72%) to cholesterol oxidase from Brevibacterium sterolicum [START_REF] Croteau | Crystallization and preliminary X-ray analysis of cholesterol oxidase from Brevibacterium sterolicum containing covalently bound FAD[END_REF].

Homologues of hsaE and hsaG have been identified in Rhodococcus sp. RHA1 and were predicted to function in cholesterol ring degradation (Table 1; [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF]). The gene hsd4B is homologous to part of the eukaryotic gene encoding the central domain of 17βHSD4, and to the hsd4B gene of Rhodococcus sp. RHA1, which is a 2-enoyl acyl-CoA hydratase, proposed to catalyze a step in the cholesterol side-chain shortening (Table 1; [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF])

The genetic organization of this cluster is (semi)conserved in several other actinomycetes, e.g. in Nocardia farcinica, Rhodococcus sp. RHA1 and Mycobacterium tuberculosis [START_REF] Mcleod | The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse[END_REF][START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF][START_REF] Ishikawa | The complete genomic sequence of Nocardia farcinica IFM 10152[END_REF], indicating a highly relevant role in cholesterol catabolism.

Heterologous expression of kstD3 in E. coli

The kstD3 gene of strain R. erythropolis SQ1 was heterologously expressed in E. coli strain BL21(DE3) for further biochemical analysis. E. coli strain BL21(DE3), containing the pET15b-KSTD3 expressed active KSTD3 at moderate levels (approx. 5% of total protein, every lane contains approx. 50 µg protein, Fig. 3) when grown for 2 days at 16

ºC. The kstD3 gene encodes a protein of 574 amino acids with a calculated molecular weight of 61 kDa, which corresponds to the band of KSTD3 on the SDS PAGE gel (Fig. 3). The expression of KSTD3 is minimal compared to expression of KSTD1 (approx.

50% of total protein) and KSTD2 (approx. 20% of total protein). Expression at higher with cell-free extracts.

KSTD3 displays 3-keto-5α-steroid ∆1-dehydrogenase activity

Based on sequence similarities with other ∆1 KSTD proteins, KSTD3 is expected to encode ∆1 KSTD activity. However, incubations of cell free extracts containing KSTD3 with 4-AD (200 µM) did not reveal detectable initial activity in a standard assay using DCPIP as artificial electron acceptor. Interestingly, ∆1 KSTD enzymes characterized thus far all display relatively high activities with 4-AD [START_REF] Itagaki | Spectral properties of 3-ketosteroid ∆ 1 -dehydrogenase from Nocardia corallina[END_REF][START_REF] Van Der Geize | Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1[END_REF][START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]7,13,[START_REF] Sih | Steroid 1-dehydrogenase of Nocardia restrictus[END_REF]. A range of steroid substrates was subsequently tested with KSTD3 (Table 2) and activity was detected with 5α-AD. To verify that KSTD3 indeed performs ∆1-dehydrogenation of 5α-AD, product formation in a standard assay was analyzed by HPLC. Incubation of KSTD3 with 5α-AD resulted in the formation of 1-(5α)-AD, the expected product of 5α-AD ∆1dehydrogenation, identified using authentic 1-(5α)-AD.

Interestingly, HPLC analysis also revealed that KSTD3 in E. coli cell-free extracts catalyzes ∆1-dehydrogenation of 5α-AD in the absence of an artificial electron acceptor, suggesting that KSTD3 uses O 2 as electron acceptor. Oxidase activity has been previously described for KSTD of Nocardia corallina [START_REF] Itagaki | Purification and characteriztion of 3-ketosteroid-∆ 1 -dehydrogenase from Nocardia corallina[END_REF]. Intriguingly, aerobic

KSTD3 displays a narrow substrate specificity compared to KSTD1 and KSTD2

Cell free extracts of E. coli Bl21(DE3), containing either KSTD1, KSTD2 or KSTD3, were used to compare the substrate specificity of KSTD3 with that of KSTD1 and KSTD2 (Table 2). KSTD activity was not observed in control cell-free extracts of E. coli Bl21(DE3) harboring the expression vector without a kstD gene. Clearly, KSTD3 has a very narrow substrate range compared to KSTD1 and KSTD2. KSTD3 activity was detected with 5α-AD, 17β-hydroxy-5α-androstane-3-one (5α-testosterone, 5α-T) and 5αpregnane-3,20-dione (5α-P), but not with 5β-H or 3-hydroxy isomers tested. KSTD enzymes require a carbonyl oxygen at C3 that interacts with an electrophilic residue, thereby facilitating the extraction of the C2(β) hydrogen as a proton by a nucleophilic residue [START_REF] Itagaki | Steroid transhydrogenase activity of 3-ketosteroid-∆1-dehydrogenase from Nocardia corallina[END_REF]. It is thought that an essential histidine is acting as a nucleophile [START_REF] Matsushita | Essential histidine residue in 3-ketosteroid-∆ 1 -dehydrogenase[END_REF].

5α-T appears to be the preferred KSTD3 substrate, with an approximately 2-fold lower K Mapp compared to 5α-AD. A 5α-H-3-keto configuration of the steroid substrate appears to be important for high KSTD3 activity. KSTD3 activity was not observed with 5αsteroids having a bulky side chain at C-17, indicating that only steroids carrying a small (5α-P), or no aliphatic side chain (5α-AD, 5α-T) are suitable substrates for KSTD3.

Contrary to KSTD1 and KSTD2, KSTD3 does not, or hardly at all, display activity with the common unsaturated ∆4 steroids (e.g. 4-AD, 9OHAD), with the exception of a minor activity observed for 4-pregnene-3,20-dione (progesterone). The 5α-3-ketosteroids 5α-AD and 5α-T are substrates for all three ∆1 KSTD enzymes studied. KSTD3 can be

S t a g e 2 ( a ) P O S T -P R I N T

distinguished from KSTD1 and KSTD2 by its preference for steroids with a saturated Aring. KSTD3 and KSTD2 both can not use NBT as an artificial electron acceptor; the unknown ∆1 KSTD activity band in Figure 1 is therefore not the KSTD3 protein characterized in this report.

The genomic co-location of kstD3 with other cholesterol catabolic genes in R.

erythropolis SQ1 (Table 1; Fig. 2 1; Fig. 2).

Phylogeny of (putative) 3-ketosteroid dehydrogenases of several actinomycetes

The genome sequences of several actinomycetes were analyzed for KSTD homologues by searching for high hits with the amino acid sequences of KSTD1, KSTD2, KSTD3 and ∆4 KSTD. Construction of a phylogenetic tree, based on the complete amino acid sequences of the different (putative) KSTDs, revealed that the ∆1 KSTDs cluster into at least four distinct groups (Fig. 4). Three of the four groups also have a ∆1 KSTD representative of R. erythropolis. Mycobacterium smegmatis has 6 putative ∆1 KSTDs [START_REF] Brzostek | Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis[END_REF], one of which does not belong to any of the groups in this phylogenetic tree (Fig. 4).

S t a g e 2 ( a ) P O S T -P R I N T 18

This suggests that the natural diversity of ∆1 KSTDs is even more elaborate than 4 groups. Among the ∆1 KSTDs, the identity of the KSTD3 amino acid sequences are slightly more conserved (64-73%) as compared to KSTD1 (40-70%) and KSTD2 (58-70%). The KSTD enzymes display significant similarity with fumarate reductases (Frd; 20-24%). Alignment of amino acid sequences of Frd of Shewanella putrefaciens with those of ∆1 KSTDs revealed four conserved regions (I-IV; Fig. 5) in addition to the conserved FAD-binding domain in the N-terminal part of the protein [START_REF] Mólnar | Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-∆ 1 -dehydrogenase, 3-ketosteroid-∆ 5 -isomerase and a hypothetical regulatory protein[END_REF][START_REF] Florin | Comamonas testosteroni 3-ketosteroid-∆ 4 -dehydrogenase: gene and protein characterization[END_REF]. Region I has been suggested to comprise the active site residues of KsdD of A. simplex, based on an alignment with the active site of Frd [START_REF] Mólnar | Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-∆ 1 -dehydrogenase, 3-ketosteroid-∆ 5 -isomerase and a hypothetical regulatory protein[END_REF]. However, our alignment indicates that the active site residues H365, E378 and R381 of Frd do not align with Region I, as was proposed for KsdD of A. simplex, but are scattered around conserved Region II. Active site residue R402 of Frd, responsible for donation of a proton to the substrate [START_REF] Leys | Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1[END_REF][START_REF] Doherty | Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study[END_REF][START_REF] Mowat | Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase[END_REF][36], is also found near region II and interestingly is occupied by a tyrosine in most KSTDs (Fig. 5). Tyrosine residues have been implicated as important for KSTD activity [START_REF] Fujii | Essential tyrosine residues in 3-ketosteroid-∆1-dehydrogenase from Rhodococcus rhodochrous[END_REF]. Residues Y104 and Y116 in KsdD of R. rhodochrous, thought to be involved in steroid substrate binding, however, are not conserved in the different types of ∆1 KSTDs. Residue Y121 in KsdD of R. rhodochrous, on the other hand, is conserved among all ∆1 KSTDs and was shown to be essential for catalysis [START_REF] Fujii | Essential tyrosine residues in 3-ketosteroid-∆1-dehydrogenase from Rhodococcus rhodochrous[END_REF]. Conserved region IV includes two key substrate-binding residues identified in Frd, i.e. H504 and R544 [START_REF] Leys | Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1[END_REF][START_REF] Doherty | Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study[END_REF][START_REF] Mowat | Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase[END_REF][36]. Residue H505 in region IV may be involved in modulation of the pK a of H504, while residues T506, M507,G508, E534 and T536 in Frd are important for coordinating a sodium ion positioned close to both the FAD-cofactor and the active site of Frd [START_REF] Pankhurst | Role of His505 in the soluble fumarate reductase from Shewanella frigidimarina[END_REF]. The threonine residue, corresponding to T506 of Frd in region IV, is fully conserved among KSTD

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enzymes and was suggested to be important for substrate binding or catalysis in KSTD2 of R. erythropolis SQ1 [START_REF] Van Der Geize | Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid delta(1)-dehydrogenase isoenzyme[END_REF]. Interestingly, the cysteine residue located next to this conserved threonine seems to be characteristic for KSTD2 enzymes.

Rv3537 of M. tuberculosis H37Rv is a KSTD3 orthologue

Remarkably, among the ∆1 KSTDs only the KSTD3 clusters with a putative KSTD of M.

tuberculosis (Fig. 4). The putative 3-ketosteroid dehydrogenase Rv3537 of M.

tuberculosis H37Rv is essential for survival in the macrophage [20]. To analyze whether the biochemical characteristics of different KSTDs in one distinct cluster are similar, the substrate profiles of Rv3537 of M. tuberculosis and KSTD3 of R. erythropolis were compared. Rv3537 from M. tuberculosis H37Rv was expressed in E. coli Bl21(DE3), using the same conditions as for KSTD3 expression. ∆1 KSTD enzyme assays and HPLC product analysis revealed a highly similar substrate range and apparent substrate affinities for Rv3537 compared to KSTD3 (Table 2). The substrate range for Rv3537 was even more limited than for KSTD3, since its ∆1 KSTD activity towards 5α-P was below detection limits.

Similarity searches and phylogenetic analysis show that Rv3537 of M. tuberculosis

H37Rv is the only ∆1 KSTD enzyme present in H37Rv [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF]. Rv3537 plays an important role in macrophage survival of M. tuberculosis [20] and in the cholesterol catabolic pathway [START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF], suggesting that 5α-AD and/or 5α-T are important steroid intermediates with respect to pathogenicity. KSTD exclusively occurs as a bacterial enzyme, i.e. there are no known human orthologues, marking KSTD as an interesting new drug target to fight tuberculosis.

  5 min 72ºC with forward primer kstD3 exp -F [5' ggcatatgagcaagcaggaatacgacatc] including start codon and NdeI restriction site (underlined) and reverse primer kstD3 exp -R [5' ggagatcttcaattggcatggttcgaccgcc] including stop codon and BglII restriction site (underlined). The resulting PCR product of kstD3 (1,725 nt) was cloned into NdeI/BamHI digested pET15b (Novagen) thereby introducing an N-terminal His-tag into the protein.

  16 ºC, 200 rpm, in LB broth supplemented with 0.5 M sorbitol and ampicillin (100 µg/ml). Isopropyl β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM at inoculation (kstD1, kstD2 expression) or after 24 h of incubation for kstD3 expression. After 24 h (kstD3) or 48 h (kstD1, kstD2) induction, cells were harvested by 9 androstane-3,17-dione (Steraloids), 1-(5α)-androstene-3,17-dione (Steraloids) (1-(5α)-AD), 11α-hydroxy-4-norandrostene-3,17-dione (Organon Biosciences), 1,4androstadiene-3,17-dione (ADD) (Organon Biosciences), dehydroisoandrosterone (Organon Biosciences), 5α-pregnan-3,20-dione (5α-P) (Steraloids), 4-cholesten-3-one (Sigma), 5α-cholestan-3-one (Acros organics, Geel, Belgium) and 5α-androstane-3β-ol, 17-one (Steraloids). Enzyme activities were measured spectrophotometrically at 30 ºC using DCPIP (extinction coefficient of 21 mM -1 cm -1 ) as artificial electron acceptor. OD 600 -measurements were followed with the "Soft-max PRO 4 (lifescience edition)" program. Curve fitting of the data was performed with the "SigmaPlot" program (version 10) using the Michaelis-Menten formula [y=(a * x)/ (b + x)]. The reaction mixture of 1 ml consisted of 50 mM TRIS pH 7.4, 80 µM DCPIP, E. coli cell-free extracts (see above) and varying concentrations of steroid substrates dissolved in ethanol (or methanol in case of 5α-pregnan-3,20-dione and 5α-cholestan-3-one). No activity was detected in control reaction mixtures lacking steroids or cell-free extracts, or extracts of E. coli with empty pET15b expression vector. Phylogenetic tree construction Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1 [26]. Amino acid sequences of different KSTD enzymes of different actinomycetes and of Comamonas testosteroni were obtained from Genbank or TIGR. The amino acid sequences (accession number between brackets) of KSTD1 (AAF19054), KSTD2 (AAL82579), KSTD3 (ABW74859) and KST4D (ABW74858) of R. erythropolis SQ1, KsdD (AB007847) of Rhodococcus rhodochrous, KsdD (BAA07186) of Arthrobacter S t a g e 2 ( a ) P O S T -P R I N T 10 simplex, MAP0530 (NP959464), MAP1433c (NP960367), MAP3398c (NP962332), MAP0696 (NP959670), MAP1530 (NP960464) and MAP1447c (NP960381) of Mycobacterium avium subsp. paratuberculosis str. k10, MSMEG5941 (YP890167), MSMEG2869 (YP887189), MSMEG4864 (YP889120), MSMEG2867 (YP887187), MSMEG4870 (YP889125), MSMEG2873 (YP887193) and MSMEG3657 (YP887960) of Mycobacterium smegmatis, Rv3537 (CAB05041) and Rv1817 (CAB01462) of Mycobacterium tuberculosis H37Rv, nfa4630 (BAD55305), nfa40910 (BAD58940), nfa22440 (BAD57091), nfa4640 (BAD55306) and nfa30830 (BAD57930) of Nocardia farcinica IFM 10152, ro05798 (YP705733), ro02483 (YP702446), ro04532 (YP704476), ro09040 (YP708242), ro09023 (YP708225), ro05813 (705748) and ro05698 (YP705635) of Rhodococcus sp. RHA1, TesI (BAC81693) and TesH (BAC81692) of Comamonas testosteroni and orf00954, orf01263, orf01472, orf01880, orf04377 and orf06377 (orf Glimmer prediction) of Rhodococcus equi were used for phylogenetic tree construction. The sequence data of Rhodococcus equi were produced by the Rhodococcus equi Sequencing Group at the Sanger Institute and were obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/re. Heterologous expression of Rv3537 of Mycobacterium tuberculosis H37Rv in E. coli cells PCR was performed on genomic DNA of M. tuberculosis H37Rv (Dick van Soolingen, RIVM, The Netherlands) using 30 cycles of 1 min 94ºC, 1 min 60 ºC and 1.5 min 72ºC, with forward primer kstD Rv3537 -F [5' cgctcgagatgactgtgcaggagttcgacg] including start codon and XhoI restriction site (underlined) and reverse primer kstD Rv3537 -R [5' cgagatcttcagcgctttcccgcctgatc] including stop codon and BglII restriction site (underlined). S t a g e 2 ( a ) P O S T -P R I N T 11 The PCR product was ligated into the XhoI/BamHI restriction site of the pET15b-vector. The pET15b-Rv3537 construct was transformed into E. coli strain BL21(DE3) resulting in strain BL21pET15bRv3537. The protein KSTD3 H37Rv was expressed under the same conditions as KSTD3 SQ1 . S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071130 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071130 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2 (

 2 a ) P O S T -P R I N T 13 characterized additional kstD genes that might be responsible for the ongoing consumption of cholesterol in the kstD1 kstD2 mutant strain RG8. Isolation and cloning of additional kstD genes from Rhodococcus erythropolis SQ1 Alignment of annotated (e.g. KSTD1, KSTD2) and putative ∆1 KSTD protein sequences identified by similarity searches in databases (http://www.ncbi.nlm.nih.gov/BLAST/) revealed the presence of conserved domains. Based on two conserved domains, the VVLAAGGF motif (a.a. 225-232 in KSTD1 SQ1 ) and GLYAAG motif (a.a. 471-476 in KSTD1 SQ1

S t a g e 2 (

 2 a ) P O S T -P R I N T 15 temperatures resulted in production of inactive KSTD3. Attempts to purify active Histagged KSTD3 protein were unsuccessful, since KSTD3 activity was lost upon purification using a Ni-NTA column. Despite exhaustive attempts, using different elution agents (e.g. NaCl, Imidazol, histidine) or additives (e.g. DTT, FAD, FMN, glycerol), we were unable to obtain purified active KSTD3 protein. Since the E. coli background lacks all KSTD activity, we decided to perform subsequent biochemical analysis of KSTD3

S t a g e 2 (

 2 a ) P O S T -P R I N T 16 dehydration by KSTD has been linked to transmembrane potential generation in Arthrobacter globiformis [29]. KSTD3 thus may have a similar function in Rhodococcus.

S t a g e 2 (

 2 a ) P O S T -P R I N T This project is financially supported by the Netherlands Ministry of Economic Affairs 420 and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private 421 NWO-ACTS program (ACTS = Advanced Chemical Technologies for Sustainability). 422 We also thank Dick van Soolingen (RIVM, The Netherlands) for kindly providing 423 genomic DNA of M. tuberculosis H37Rv and Organon Biosciences for supporting this 424 project. 425 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071130 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071130 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 Gene cluster and their proposed role in cholesterol degradation

Figure 3

 3 Figure 3 Expression of KSTD enzymes in E.coli cell free extract

Figure 4 Figure 5

 45 Figure 4 Phylogenetic tree showing different KSTD enzyme clusters

S t a g e 2 ( 32 Figure 1 ∆1Figure 2 S t a g e 2 ( 34 Figure 3 35 Figure 4 S t a g e 2 (

 2321223433542 Figure 1

  ), as well as strong up-regulation of its homologue in cholesterol grown cells of Rhodococcus sp. RHA1[START_REF] Van Der Geize | A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages[END_REF], indicate a role for KSTD3 in cholesterol degradation. Unexpectedly, the preferred substrate of KSTD3 is not 4-AD, the commonly accepted central intermediate in sterol/steroid degradation, but the 5α-reduced

form of 4-AD. The data thus suggest that 5α-T and 5α-AD are pathway intermediates preceding 1,4-androstadiene-3,17-dione (ADD) during cholesterol catabolism. The conversion of 5α-AD into ADD requires the action of a ∆1 KSTD, encoded by kstD3, and a ∆4 KSTD, putatively encoded by kst4D. Both activities appear to be encoded by the cloned DNA fragment (Table

Table 1 Isolated gene cluster of Rhodococcus eryrthropolis SQ1 and their 630 homologues 631

 1 Homologues of the 6 Open Reading Frames (ORF) of R. erythropolis SQ1 located on 632 pJK2 and the (putative) functions of the proteins encoded. 633

	Gene	Annotation	Annotated best hit (% identity)	Reference
	kst4D	3-ketosteroid-delta4(5alpha)-dehydrogenase	TesI of Comamonas testosteroni (45%)	[12]
	kstD3	3-ketosteroid-delta-1-dehydrogenase	KSTD (ro04532) of Rhodococcus sp. RHA1 (78%)	[19]
	hsaG	Acetaldehyde dehydrogenase	HsaG (ro04534) of Rhodococcus sp. RHA1 (96%)	[19]
	hsaE	2-hydroxypentadienoate hydratase	HsaE (ro04533) of Rhodococcus sp. RHA1 (87%)	[19]
	choG	Cholesterol oxidase	BCO2 of Brevibacterium sterolicum (72%)	[27]
	hsd4B	2-Enoyl acyl-CoA hydratase	HSD4B (ro04531) of Rhodococcus sp. RHAI (83%)	[19]

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071130 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071130

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 Substrate profiles of KSTD1, KSTD2 and KSTD3 of R. erythropolis SQ1, 634 and KSTD3 of M. tuberculosis H37Rv, expressed in cell-free extracts of E. coli 635

 2

	636	Substrate profiles of KSTD1, KSTD2 and KSTD3 of R. erythropolis SQ1, and KSTD3
	637	of M. tuberculosis H37Rv, expressed in cell-free extracts of E. coli Bl21(DE3)
	638	determined under standard assay conditions (see experimental procedures). Enzyme
	639	activities (V max ) were calculated in nmol -1 •min -1 •mg -1 total protein in CFE and are
	640	expressed as percentage compared to activity with 5α-androstane-3,17-dione which was
	641	set at 100% (2328, 860, 216, 178 are the 100% values of KSTD1, KSTD2, KSTD3 SQ1
	642	and KSTD3 H37Rv , respectively). K Mapp indicates the apparent affinity constants in µM.
	643	Values shown are ± SEM (n=3). n.d.: enzyme activity was below detection limits. Note
	644	that the low solubility of steroids may affect measurements, therefore the kinetic
	645	parameters do not represent absolute but relative values, providing a strong indication of
	646	the substrate preference of the KSTD enzymes.
			KSTD1	KSTD2	KSTD3 SQ1	KSTD3 H37Rv
	Substrate	Rel.V max	K Mapp	Rel.V max	K Mapp	Rel.V max	K Mapp	Rel.V max	K Mapp
	17β-hydroxy-5α-androstane-3-one (5α-T)	84% ± 3	36 ± 4	59% ± 3	6 ± 1	97% ± 10	36 ± 3	73% ± 14	33 ± 10
	5α-androstane-3,17-dione (5α-AD)	100%	59 ± 3	100%	13 ± 3	100%	70 ± 12	100%	166 ± 16
	5β-androstane-3,17-dione	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	4-androstene-3,17-dione (4-AD)	574% ± 14	104 ± 5	249% ± 7	5 ± 1	n.d.	n.d.	n.d.	n.d.
	9α-hydroxy-4-androstene-3,17-dione (9OHAD)	655% ± 30	119 ± 11	186% ± 8	58 ± 9	n.d.	n.d.	n.d.	n.d.
	1-(5α)-androstene-3,17-dione (1-(5α)-AD)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	5α-androstane-3β-ol, 17-one	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	1,4-androstadiene-3,17-dione (ADD)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	Progesterone (P)		340% ± 8	18 ± 2	460% ± 6	3 ± 1	25% ± 3	29 ± 6	35% ± 6	65 ± 4
	5α-pregnan-3,20-dione (5α-P)	32%± 2	21 ± 5	12%± 1	7 ± 1	47% ± 8	14 ± 4	n.d.	n.d.
	23,24, bisnor 5α-cholestane-3-one acid	69% ± 2	74 ± 4	60%± 2	26 ± 4	n.d.	n.d.	n.d.	n.d.
	11α-hydroxy-19-norandrost-4-ene-3,17-dione	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	11β-hydrocortisone		88% ± 14	557 ± 120	70% ± 2	64 ± 5	n.d.	n.d.	n.d.	n.d.
	4-cholesten-3-one		n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	5α-cholestan-3-one		n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	Dehydroisoandrosterone	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071130 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071130 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071130 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society


Hua Ren

Shuo-Xing Dou

Xing-Dong Zhang

Peng-Ye Wang

Radhakrishnan Kanagaraj

Pavel Janscak

Jin-Shan Hu
email: jshu@westernu.edu

Xu Guang Xi

Xu Guang Xi
email: guang.xi@curie.u-psud.fr

The zinc-binding motif of human RECQ5β suppresses the intrinsic strand annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme

Keywords: RecQ helicase, DNA unwinding, DNA annealing, zinc-finger motif, intramolecular regulation

come

The zinc-binding motif of human RECQ5β suppresses the intrinsic strand annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme

INTRODUCTION

DNA helicases are molecular motors that transduce the chemical energy derived from NTP hydrolysis into mechanical force to unwind double-stranded DNA (dsDNA) [START_REF] Lohman | Mechanisms of helicase-catalyzed DNA unwinding[END_REF][START_REF] Soultanas | DNA helicases: 'inching forward[END_REF]. Therefore, helicases display both DNA-stimulated ATPase and ATP-dependent helicase activities. Structural studies revealed that helicases from superfamily 1 and 2 (SF1 and SF2) contain a structural module that consists of two RecA-related domains [START_REF] Caruthers | Helicase structure and mechanism[END_REF][START_REF] Singleton | Modularity and specialization in superfamily 1 and 2 helicases[END_REF]. Residues involved in ATP binding/hydrolysis and DNA binding are located in the cleft separating the two RecA-like domains. It is postulated that these domains serve as a DNA translocation motor [START_REF] Singleton | Modularity and specialization in superfamily 1 and 2 helicases[END_REF].

The RecQ family DNA helicase are highly conserved from bacteria to man and play essential roles in the maintenance of genomic stability [START_REF] Yamagata | Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases[END_REF][START_REF] Hanada | RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli[END_REF][START_REF] Hickson | RecQ helicases: caretakers of the genome[END_REF][START_REF] Mankouri | Understanding the roles of RecQ helicases in the maintenance of genome integrity and suppression of tumorigenesis[END_REF]. In humans, five RecQ family members, namely RECQ1 [START_REF] Puranam | Cloning and characterization of RECQL, a potential human homologue of the Escherichia coli DNA helicase RecQ[END_REF][START_REF] Seki | Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12[END_REF], BLM [START_REF] Ellis | The Bloom's syndrome gene product is homologous to RecQ helicases[END_REF], WRN [START_REF] Yu | Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes[END_REF], RECQ4 [13] and RECQ5 [START_REF] Shimamoto | Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta[END_REF], have been identified. Defects in BLM, WRN and RECQ4 lead to human hereditary disorders: Bloom syndrome (BS), Werner syndrome (WRN) and Rothmund-Thompson syndrome (RTS), respectively, that are characterized by genome instability and cancer predisposition [START_REF] Karow | RecQ family helicases: roles in cancer and aging[END_REF]. In addition to the highly conserved DExH helicase domain containing seven helicase motifs, most RecQ family helicases contain a unique RQC domain (RecQ conserved) which is composed of a zinc-binding motif and a winged helix-turn-helix (WH) motif [START_REF] Morozov | A putative nucleic acid-binding domain in Bloom's and Werner's syndrome helicases[END_REF][START_REF] Bernstein | High-resolution structure of the E. coli RecQ helicase catalytic core[END_REF]. RecQ helicase activity is highly regulated not only through inter-molecular protein-protein interactions [START_REF] Bachrati | RecQ helicases: suppressors of tumorigenesis and premature aging[END_REF], but also through intra-molecular interactions between different domains [START_REF] Hu | Solution structure of a multifunctional DNA-and protein-binding motif of human Werner syndrome protein[END_REF]. In this study, we have chosen the human RECQ5 protein as a model to study the functional modulation of the DExH-helicase core by the zinc-binding domain. The striking feature of human RECQ5 is that it exists in three different isoforms, namely RECQ5α, RECQ5β and RECQ5γ, which result from alternative splicing of the RECQ5 transcript [START_REF] Shimamoto | Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta[END_REF][START_REF] Sekelsky | Drosophila and human RecQ5 exist in different isoforms generated by alternative splicing[END_REF]. The three proteins are identical within the N-terminal region of 410 residues which constitutes the helicase domain with the conserved set of seven helicase motifs (Supplementary Figure 1). RECQ5α is the shortest variant including only the helicase domain (410 amino acids). RECQ5β contains the helicase domain and a putative RQC domain, which is followed by a long C-terminal region that shows no homology to the other RecQ helicases.

RECQ5γ includes the helicase domain followed by a C-terminal extension of 25 amino acids that is not present in RECQ5β. At present, only RECQ5β has been characterized biochemically [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]. It possesses both DNA-unwinding and DNA strand-annealing activities [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]. In addition, RECQ5β has been shown to promote strand exchange on forked DNA structures [START_REF] Kanagaraj | Human RECQ5beta helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork[END_REF]. However, the molecular mechanism that underlies the coordination of DNA unwinding and strand-annealing activities of RECQ5β remains largely elusive.

The RECQ5α and RECQ5β proteins offer an attractive model system to characterize the enzymatic activities of a RecQ helicase domain in isolation and in association with other sub-domains on the same polypeptide. This unique property allows us to address some interesting and important questions that have not yet been resolved with any DNA helicase. These questions include : (i) Is the helicase domain sufficient to catalyze dsDNA separation;

(ii) Does the helicase domain have an intrinsic strand-annealing activity as suggested by its structural similarity to the strand-exchange protein RecA; (iii) What is the role of the accessory domains, namely the zinc-binding domain, in the helicase activity of the enzyme. In the present study, we found that the helicase domain of RECQ5 does not possess DNAunwinding capability, but exhibits an intrinsic strand-annealing activity. More importantly, the zinc-binding motif is found to act as a molecular switch that suppresses the strandannealing activity of the helicase domain and triggers DNA-unwinding activity through modulating DNA binding.

MATERIALS AND METHODS

Proteins and DNA substrates

The human RECQ5α and RECQ5β proteins were produced as C-terminal fusions with a self-cleaving chitin-binding domain and purified as previously described [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]. The truncation mutants of RECQ5β were amplified by PCR and cloned into pET15b as N-terminal fusions with a hexa-histidine tag. The resulting plasmids were transformed into the E. coli strain BL21-codonPlus (Stratagene). The cells were grown to the mid-exponential phase (A 600 of 0.5-0.6) at 37 °C and the protein expression was induced by 0.25 mM of isopropyl-Dthiogalactoside at 15 °C for 18 h. The cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.1% Triton X-100, PMSF 0.1µM and 10% ethylene glycerol. The cell lysate was clarified by centrifugation and the supernatant was applied to a 20 ml Ni 2+column connected to an ÄKTA FPLC system. The bound proteins were eluted with a linear gradient of imidazole (0.02-0.4 M; 300 ml). Fractions containing the proteins of interest were identified by SDS-PAGE. Proteins were further purified using size-exclusion chromatography (Superdex 200, Amersham). Human replication protein A (RPA) protein was produced and purified according to previously published procedure [START_REF] Henricksen | Recombinant replication protein A: expression, complex formation, and functional characterization[END_REF].

PAGE purified DNA oligonucleotides (Supplementary Table 1) were purchased from Proligo. The DNA duplex substrates were prepared as described previously [START_REF] Dou | The DNA binding properties of the Escherichia coli RecQ helicase[END_REF]. Briefly, 250 µM component oligonucleotides were denatured in the 1× TE buffer containing 1 M NaCl or

KCl by heating at 95 °C for 10 min. The denatured DNA was then annealed at 37 °C for 48 h.

The annealed products were separated on 8% native PAGE containing 10 mM KCl run at 4

°C for 12 h with constant current of 20 mA.

Quantification of protein-bound Zn 2+ ion

Protein-bound Zn 2+ ion was measured using 4-(2-pyridylazo)resorcinol (PAR), a reporter dye that absorbs light at 490 nm when bound to Zn 2+ . To precisely quantify the Zn 2+ content, all buffers were treated with Chelex ® -100 resin (Bio-Rad). The proteins were dialysed against the EDTA-free Chelex ® -treated buffer, passed over a 10-cm column of This measurement was useful for confirming the overlap of the fluorescence emission spectra of the proteins and the excitation spectrum of mantATP near 350 nm. When excited at 280 nm, this overlap would result in, due to FRET, an enhancement of the emission of mantATP at 440 nm after its binding to the proteins.

The mantATP binding assay was performed by using a Bio-Logic auto-titrator (TCU-250) and a Bio-Logic optical system (MOS450/AF-CD) in fluorescence mode. Varying amounts of mantATP were added to 1 ml of binding buffer containing 0.5 µM protein. After 1 minute incubation, fluorescence at 440 nm was measured. Titrations were performed in a temperature-controlled cuvette at 25 °C. The solution was stirred continuously by a magnetic stir bar during the whole titration process. The apparent dissociation constant K d was determined by fitting the fluorescence intensity at 440 nm (corrected for the inner filter effect)

with Equation 1,

2 ( 0.5 ) ( 0.5 ) 4 0.5 2 d d d d d s d d c x c K x c K c x F F c f x f + + - + + -× × = + + , ( Equation 1)
where, F s is the starting fluorescence of the reaction mixture, f d is the fluorescence coefficient of free mantATP, f c is the fluorescence coefficient of the complex formed, x is the total concentration of mantATP.

( )

0 0 1 [mantATP] i d c V V V x = + ≡ - is included to correct
accurately for the sample dilution effect, where V 0 is the initial sample volume, V i is the volume of titrant added, and [mantATP] is the mantATP concentration of the titrant.

ATPase assay

The ATPase activity of the RECQ5 proteins was determined by measuring the concentration of inorganic phosphate produced by ATP hydrolysis [START_REF] Guo | Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase[END_REF]. The reaction was carried out in ATPase reaction buffer (50 mM Tris-HCl, pH 8.0, 3 mM MgCl 2 , 0.5 mM DTT) at 37 °C in a volume of 100 µl. The reactions were initiated by the addition of enzyme into a reaction mixture containing 1.5 µM heat-denatured Hind III-cut pGEM-7Zf linear DNA and the mixture of ATP and [γ -32 P]ATP. The reaction was stopped by adding 0.2 ml of the stopping buffer containing 8.1 mM ammonium molybdate and 0.8 M HCl. The liberated 32 P i was extracted with a solution of 2-butanol/benzene/acetone/ammonium molybdate (750:750:15:1) saturated with water. An aliquot of 60 µl was removed from the organic phase and the radioactivity was quantified using a liquid scintillation counter.

DNA binding assays

DNA binding of RECQ5 proteins was analyzed by fluorescence polarization anisotropy assay as described previously [START_REF] Dou | The DNA binding properties of the Escherichia coli RecQ helicase[END_REF]. The DNA binding assay was performed using Stopped-flow DNA unwinding assay was performed according to Zhang et al. [START_REF] Zhang | Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1[END_REF].

Briefly, the experiment was carried out using a Bio-logic SFM-400 mixer with a 1.5 mm× 1.5 mm cell (Bio-Logic, FC-15) and a Bio-Logic MOS450/AF-CD optical system equipped with a 150-W mercury-xenon lamp. Fluorescein was excited at 492 nm, and its emission was monitored at 525 nm. The unwinding assay was performed in a two-syringe-mixing mode,

where the protein and dsDNA substrates were preincubated in syringe #1 for 5 min, while ATP was present in syringe #4. Both syringes contained the unwinding reaction buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl 2 and 0.1 mM DTT). The unwinding reaction was initiated by a rapid mixing of the contents of the two syringes. The dsDNA substrates had both strands labeled with fluorescein and hexachlorofluorescein, respectively, and their sequences are listed in the Supplementary Table 1. During the unwinding process, the fluorescein emission signal at 525 nm was enhanced due to the loss of FRET, i.e., the separation of the two dye molecules on the two ssDNA. For converting the output signal change from volts to percentage of unwinding, a calibration experiment was performed in a four-syringe-mixing mode, where the helicase in syringe #1, hexachlorofluorescein-labeled ssDNA in syringe #2, fluorescein-labeled ssDNA in syringe #3, and ATP in syringe #4, all incubated in unwinding reaction buffer. The fluorescent signal of the mixed solution from the four syringes corresponded to 100% unwinding. The standard reaction was carried out at 25 °C.

DNA strand-annealing assay

The DNA annealing activity of the RECQ5 isoforms was assayed using complementary synthetic oligonucleotides (0.5 nM) with one strand 5′-end labelled using [γ-32 P]ATP and T4 polynucleotide kinase. In this assay, the labelled oligonuclotide was added to reaction buffer (20 µl) containing 20 mM Tris-acetate, pH 7.9, 50 mM KOAc, 10 mM Mg(OAc) 2 , 1 mM DTT, 50 µg/ml BSA and the indicated protein concentration. Where required, ATP, ATPγS, AMPPNP or ADP were also added to a final concentration of 2 mM. The reaction was initiated by adding the unlabelled oligonuclotide and incubated at 37 °C for 30 minutes. The resulting DNA products were analysed as described for the helicase reactions.

The stopped-flow DNA strand-annealing assay was carried out under similar conditions as the helicase assay. The reaction was performed in a three-syringe-mixing mode,

RECQ5 helicase domain alone catalyzes strand annealing, but not strand separation

To analyze the function of an isolated helicase domain of RecQ DNA helicases, we investigated the biochemical properties the human RECQ5α protein. We first compared the unwinding activities of RECQ5α and RECQ5β using gel-shift-based helicase assay. The results showed that RECQ5β efficiently unwound a partial DNA duplex in a dose-dependent manner, whereas RECQ5α displayed hardly detectable helicase activity, even in the presence of human RPA which was reported to stimulate RECQ5β-mediated DNA unwinding [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF] (Figure 1A and data not shown). To confirm this result, we employed a FRET-based helicase assay, which allowed the helicase-mediated DNA unwinding to be monitored in real time in a stop-flow experiment [START_REF] Zhang | Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1[END_REF]. Consistent with earlier studies, RECQ5α displayed non-detectable helicase activity, whereas RECQ5β unwound the DNA substrate efficiently (Figure 1B).

Under multiple-turnover conditions, the unwinding kinetics of RECQ5β was biphasic. The fitting using a sum of two exponential terms gave two rate constants of 4.54 and 0.84 min -1 for the fast and slow phases, respectively (Figure 1B and Table 1).

Next, we tested RECQ5α and RECQ5β for the ability to promote strand-annealing using two complementary 50-mer oligonucleotides. Remarkably, RECQ5α was found to promote strand annealing as efficiently as RECQ5β (Figure 2A). This result was rather surprising since the strand-annealing activity of RECQ5β was shown to reside in the Cterminal portion of the RECQ5β polypeptide [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]. We then examined the effect of ATP on the annealing activities of the RECQ5 proteins. Consistent with the previously published results, we found that ATP had no effect on the annealing activity of RECQ5β, whereas ATPγS, a poorly hydrolysable analogue of ATP, dramatically suppressed this reaction (Figure 2B, lanes 2 and 3). In contrast, ATP dramatically inhibited the annealing activity of RECQ5α as it did ATPγS (Figure 2B, lanes 8 and 9). We also examined the effect   of ADP on the strandannealing activities RECQ5α and RECQ5β. We found that ADP partially inhibited the annealing activity of RECQ5β, but had no effect on the annealing activity of RECQ5α (Figure 2B, lanes 4 and 10). Since previous studies showed that RPA efficiently inhibited the strand-annealing activities of RECQ5β and RECQ1 [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]27], we also tested the effect of this single-strand binding protein on the strand-annealing activity of RECQ5α. We found that similar to RECQ5β, RECQ5α-mediated strand annealing activity was inhibited by RPA in a dose-dependant manner (Figure 2C). FRET-based assay was used, which was essentially identical to the stop-flow helicase assay described above. The only difference was that the annealing assay monitored the decrease, instead of the increase, in the fluorescence emission due to FRET (Figure 2D). The measured annealing reaction rates were 0.95 min -1 and 0.14 min -1 for RECQ5β and RECQ5α, respectively. Thus, RECQ5β promoted strand annealing about seven-fold faster than RECQ5α

(Figure 2E). However, when evaluated by the extent of annealing, RECQ5α was more active than RECQ5β (Figure 2E and Table 1).

In order to understand the molecular basis of the RECQ5α-mediated strand annealing, we studied the quaternary structure of RECQ5α in the absence and in the presence of ATP and/or ssDNA by means of size-exclusion chromatography on a Superdex TM 75 column.

These experiments (see supporting information (SI) Table 2) revealed an apparent molecular mass of 50~58 kDa at all condition tested, indicating that RECQ5α promotes strand annealing as a monomer (the predicted molecular mass of monomeric RECQ5α is 46.3 kDa).

RECQ5 helicase domain binds ATP efficiently, but displays a poor ATPase activity

To understand why RECQ5α does not catalyse DNA unwinding, we analyzed its ATPbinding and ATPase activities relative to RECQ5β. To quantify ATP-binding of RECQ5α and RECQ5β, mantATP, a fluorescent analogue of ATP, was used. The apparent dissociation constants (K d ) measured were 48.4 ± 2.5 and 51.0 ± 4.9 µM for RECQ5α and RECQ5β, respectively (Figure 3A and Table 1), indicating that the two isoforms bind ATP with a similar affinity.

The ATPase activities of the RECQ5 proteins were determined by measuring the release of inorganic 32 P i from ATP in the presence of denatured dsDNA (2.9 kb) (Figure 3B).

In agreement with previously published data [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF], RECQ5β was found to exhibit a robust ATPase activity, with a k cat of 15.6 ± 1.4 sec -1 . However, under identical conditions, RECQ5α had no measurable ATPase activity (Figure 3B and Table 1).

Thease data indicate that the inability of RECQ5α to unwind DNA is caused by its failure to hydrolyze ATP. Moreover, these data provide explanation for the differential effect of ATP on the strand-annealing activities of RECQ5α and RECQ5β (Figure 2B). As

RECQ5α can not hydrolyze ATP, it would persist in ATP-bound form, which is not proficient in strand annealing [START_REF] Garcia | Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide[END_REF]. In the case of RECQ5β, this inhibitory effect can be seen only with poorly hydrolysable ATPγS.

Zinc-binding motif is essential for the helicase activity of RECQ5β

The observation that the isolated helicase domain of RECQ5β failed to unwind dsDNA suggested a role for a C-terminal part of RECQ5β in performing its helicase function.

Therefore, C-terminal truncation mutants of RECQ5β , including RECQ5β 1-475 and RECQ5β 1- 662 , were assayed to identify the C-terminal region responsible for the intramolecular stimulation of the helicase activity of RECQ5β. We found that these two mutants had essentially identical ATPase and DNA unwinding activities with respect to the full-length RECQ5β protein (Figure 3B, Figure 5 and Table 1). Together, these observations suggest that the region of RECQ5β spanning the amino acid residues 410-475 that constitute the putative zinc-binding domain (Figure 4A) plays an essential role in the ATPase and helicase activities of the enzyme. Molecular modelling of the zinc-binding motif of RECQ5β revealed that the distances between the cysteine side-chain sulphur groups and the zinc atom for Cys 411 ± 0.09 Å) for structural metal zinc-coordination sites [START_REF] Alberts | Analysis of zinc binding sites in protein crystal structures[END_REF] (Figure 4B). In agreement with this prediction, using PAR assay, we found that RECQ5β contains 0.98 ± 0.18 equivalent Zn 2+ ion (Figure 4C and Table 1). The similar results were obtained for RECQ5β 1-475 and RECQ5β 1-662 deletion mutants (Table 1). In contrast, the PAR assay indicated that RECQ5α did not contain any Zn 2+ ion, with measured Zn 2+ value of 0.03 ± 0.02 (Table 1).

To further evaluate the role of the zinc-binding motif in the helicase function of RECQ5β, we generated a panel of mutants in which one or more of the four conserved cysteine residues were replaced by serine. However, these mutant proteins either formed aggregates or were degraded except for the C431S mutant, which was subjected to biochemical analysis. We found that the replacement of Cys 431 with Ser reduced the proteinbound Zn 2+ to 0.35 ± 0.12 equivalents (Table 1). Accordingly, this mutant had a significantly reduced ATPase and helicase activities relative to wild-type RECQ5β (Figure 1B, Figure 3B and Table 1). Taken together, these data suggest that the zinc-binding motif modulates the enzymatic activity of RECQ5β: it suppresses the strand-annealing function of the helicase domain and promotes strand separation. determined from protein titration curves, indicated that RECQ5α displayed much lower binding affinities for ss-and dsDNA than RECQ5β (Figure 5 and Table 1).

Molecular mechanism of intra-molecular stimulation of the helicase activity of RECQ5 by the zinc-binding motif

It has been shown that ATP binding stimulates DNA binding of some helicases, which in turn triggers ATP hydrolysis [START_REF] Wong | Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding[END_REF][START_REF] Jezewska | Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding. Multiple conformational states of the helicase hexamer[END_REF][START_REF] Jezewska | Interactions of Escherichia coli replicative helicase PriA protein with single-stranded DNA[END_REF][START_REF] Bujalowski | Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues[END_REF]. Therefore, we measured the DNA binding activities of RECQ5 proteins in the presence of a non-hydrolyzable ATP analogue, AMPPNP and/or ATPγS. We found that the ssDNA binding affinity of RECQ5β was enhanced about 2-3 fold in the presence of 2 mM AMPPNP, whereas no noticeable effect could be observed with RECQ5α, even in the presence of ATP (ATP cannot be hydrolyzed by RECQ5α) (results not shown). These data imply that in RECQ5α , there is no co-operativity between the ATP and DNA-binding sites.

To further explore the role of the zinc-binding motif in DNA binding by RECQ5β, the RECQ5β 1-475 and RECQ5β 1-662 truncation mutants were assayed for their DNA-binding activities. We found that these two mutants had similar DNA-binding affinities as the wildtype RECQ5β protein, suggesting that the non-conserved C-terminal region of RECQ5β does not play a role in DNA binding by the enzyme and that the zinc-binding motif confers the DNA-binding proficiency to the RECQ5β helicase core (Figures 5A,5B and Table 1).

To determine whether the zinc-binding motif of RECQ5β is directly involved in DNA binding, we sought to examine the DNA binding properties of the isolated zinc-binding domain. Our first attempts to generate a fragment including only the zinc-binding fold (RECQ5β 379-454 ) failed because this protein was found to be rapidly degraded. We then performed DNA binding experiments with RECQ5β 379-662 and RECQ5β 454-662 that could be purified to homogeneity. We found that RECQ5β 379-662 , which contains the intact zincbinding motif, was bound to DNA showing a preference for dsDNA (K d values of 149±6.7 nM and 680±63 nM for dsDNA and ssDNA, respectively). In contrast, RECQ5β 454-662 with a truncated zinc-binding motif displayed hardly detectable DNA binding activity (Figures 5C and5D). Together, these experiments revealed that the zinc-binding domain enhances the DNA-binding affinity of the RECQ5β helicase core through direct DNA binding. helicases in DNA unwinding (Figure 6). Crystal structures of several DNA helicases including the E. coli RecQ helicase core suggested that ATP is bound at the top of domain 1A, while the ssDNA substrate is bound at the bottom of domains 1A and 2A (Figure 6).

Model for the role of zinc-binding motif in the function of RecQ helicases

Since more residues in domain 1A are implicated in DNA binding than in domain 2A, as revealed by the crystal structures of NS3 helicase [START_REF] Kim | Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding[END_REF] and DEAD-Box helicase Vasa [START_REF] Sengoku | Structural Basis for RNA Unwinding by the DEAD-Box Protein Drosophila Vasa[END_REF], it is likely that DNA may be bound more tightly to domain 1A than to domain 2A.

Consequently, the portion of DNA that is bound to domain 2A may undergo large conformational fluctuation. In this case, the distance between the two helicase domains is too far to interact with each other to form the functional ATPase site that is essential for DNA unwinding. However, it is conceivable that while domain 1A binds ssDNA tightly, domain 2A can capture another ssDNA molecule. Thus, RECQ5α would function as a 'molecular crowding agent' [START_REF] Minton | Influence of excluded volume upon macromolecular structure and associations in 'crowded' media[END_REF] that increases the effective concentrations of ssDNA substrates, thereby promoting the hybridization between complementary ssDNA molecules (Figure 6). However, in the presence of the zinc-binding motif, the double-stranded part of the DNA substrate would be tightly bound to the enzyme, enhancing the ssDNA binding to domain 2A (Figure 6). The tight binding of DNA to domain 1A and 2A, in collaboration with ATP binding, brings the domains into the closed form. In this structure, ATP is efficiently hydrolyzed and the energy is coupled to DNA unwinding.

Concluding remarks

The zinc-binding motif conserved among the RecQ family helicases does not have substantial structural similarity with any other known zinc-finger folds. It is therefore important to determine its specific function in the RecQ helicase family. We have previously shown that the zinc-binding motifs of E. coli RecQ and human BLM are important for DNA binding and protein folding of these helicases [START_REF] Guo | Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase[END_REF][START_REF] Liu | The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding[END_REF]. Thus, there is a clear correlation between the presence of the zinc-binding motif and the helicase activity of RecQ helicases.

Interestingly, the human RECQ4 protein, the only RecQ family member without zinc-binding motif, fails to unwind DNA, but catalyzes DNA annealing, which is in agreement with our data obtained with RECQ5α [START_REF] Macris | Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome[END_REF]. Thus, all of these studies establish the RecQ-specific zincbinding motif as an essential DNA-binding module required for the RecQ family helicase activity. Hill equations. The apparent K d values are summarized in Table 1.

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  Chelex ® -100 and re-concentrated. To facilitate the Zn 2+ release, the protein (about 1 nmole in 40 µl) was first denatured with Chelex ® -treated 7 M guanidine-HCl, and then transferred to a 1-ml cuvette. PAR was added to the cuvette to a final concentration of 100 µM and the volume was adjusted to 1 ml with PAR buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The absorbance was recorded from 300 to 600 nm on an UVIKON Biochemical Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T 6 spectrophotometer 941 (Kontron) at 25 °C. The quantity of Zn 2+ was determined using the absorbance coefficient for the Zn (PAR) 2 complex (ε 500 = 6.6×10 4 M -1 cm -1 ). As a control, 20 nmoles of the pure ZnCl 2 was quantified under the identical conditions. ATP binding assay 2'(3')-O-(N-methlanthraniloyl) adenosine-5'-triphosphate, a fluorescent nucleotide analogue of ATP (mantATP), was used to determine the apparent dissociation constant for ATP binding by RECQ5 proteins. For that purpose, the fluorescence spectra of the proteins at a concentration of 0.5 µM were first measured using FluoroMax-2 spectrofluorimeter (Jobin Yvon, Spex Instruments S.A., Inc) at 25 °C in a 10×10×40 mm quartz cuvette. The proteins were excited at 280 nm and their intrinsic fluorescence emission near 350 nm was monitored.

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the

  Bio-Logic auto-titrator (TCU-250) and the Bio-Logic optical system (MOS450/AF-CD) in a fluorescence anisotropy mode. Varying amounts of proteins were added to 1 ml of binding buffer containing 1 nM DNA substrate. After 1-minute incubation, the fluorescence polarization anisotropy was measured. The temperature was controlled at 25 °C and the solution was stirred continuously during the whole titration process. The equilibrium dissociation constant was determined by fitting the data to the Michaelis-Menten or Hill equation using the program KaleidaGraph (Synergy Software).DNA helicase assaysDNA unwinding reactions were carried out at 37 °C in 20 µl of the mixture containing 25 mM HEPES-NaOH, pH 7.5, 25 mM NaOAc, 7.5 mM Mg(OAc) 2 , 2 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, and indicated 32 P-labelled partial DNA duplex substrate (10 fmol, 3000 c.p.m/fmol). Where required, RPA was added at the indicated concentration. The reaction was initiated by addition of the indicated concentration of the RECQ5 protein and incubated at 37 °C for 30 minutes. Reactions were terminated by the addition of 5 µl of 5× loading buffer (50 mM EDTA, 0.5% SDS, 0.1% xylene cyanol, 0.1% bromophenol blue and 50% glycerol). The reaction products were resolved on a 12% (w/v) PAGE run in a TBE buffer (90 mM Tris, 90 Biochemical Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T mM boric acid, pH 8.3, and 1 mM EDTA) at 100 V for 2 h at 4 °C. Radiolabelled species were visualized using a Storm 860 Phosphoimager (Amersham Biosciences).

  Biochemical Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T where the helicase was in syringe #1 while fluorescein-and hexachlorofluorescein-labeled complementary ssDNA in syringe #2 and syringe #3, respectively, in buffer containing 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl 2 and 0.1 mM DTT. The sequences of the two 45-mer ssDNA substrates are indicated in the Supplementary Table 1. The reaction was initiated by rapidly mixing the contents of the three syringes. During the annealing process, the fluorescein emission signal at 525 nm was reduced due to FRET from fluorescein to hexachlorofluorescein on the two ssDNA. For converting the output signal change from volts to percentage of annealing, we performed another experiment in a two-syringe-mixing mode, where the helicase was in syringe #1 and the annealed dsDNA in syringe #4, both in the same buffer as indicated above. The fluorescent signal of the mixed solution from the two syringes corresponded to 100% annealing. The reaction was carried out 25 °C. Biochemical Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T 10 RESULTS AND DISCUSSION

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  Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T 11 To quantitatively compare the DNA annealing kinetics of the RECQ5 isoforms, a

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Biochemical

  Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T 13 In order to understand the molecular mechanism underlying the essential role of the zinc-binding motif in the helicase activity of RECQ5β, we compared the DNA binding properties of RECQ5α and RECQ5β using fluorescence polarization assay with fluorescentlylabelled oligonucleotide substrates [24]. This method allows direct measuring of DNA binding in solution under equilibrium conditions. K d values for RECQ5α and RECQ5β

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2008 as manuscript BJ20071150 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071150 S t a g e 2 ( a ) P O S T -P R I N T 14 Based on the results presented above in combination with structural information for SF2 helicases, we proposed a model for the function of the zinc-binding motif of RecQ DNA

Biochemical

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Figure 1

 1 Figure 1 Helicase assays of RECQ5α and RECQ5β (A) Gel-based assay of DNA unwinding using 0.5 nM [5'-32 P]-labelled 23-base DNA duplex (UA/UB). Reactions were carried out at 37 °C for 30 minutes under condition as described in Materials and Methods. The reaction products were analyzed by 12% non-denaturing PAGE. (B) Stopped-flow DNA unwinding assay. As described in Materials and Methods, 1 nM 56:16-mer DNA substrate (UFA/UFB) was preincubated with 20 nM helicase for 5 min at 25 °C. Unwinding reaction was initiated by mixing with 1 mM ATP. The fluorescence emission of fluorescein at 520 nm (excited at 492 nm) was monitored. The increase of the fluorescence signal of fluorescein at 525 nm (excited at 492 nm) during unwinding was monitored. Fraction of DNA unwound was obtained by normalization of the fluorescence signal F using the value F max as obtained from the calibration measurement that corresponded to 100% unwinding (see Materials and Methods), i.e., Fraction unwound = (F-F s )/( F max -F s ), where F s is the fluorescence signal at the start of unwinding.

Figure 2 RECQ5

 2 Figure 2 RECQ5-mediated ssDNA annealing (A) Annealing of the [5'-32 P]-labelled SPA (0.5 nM) and SPB ssDNA substrates by RECQ5α and RECQ5β. Reactions were incubated with the indicated protein concentrations for 30 min at 37 °C. The reaction products were separated by 12% non-denaturing PAGE and visualized by PhosphoImager. (B) Effect of nucleotides on the RECQ5-mediated ssDNA annealing. The reactions contained 50 nM RECQ5α or RECQ5β and were carried under the same conditions as in (A). Preformed 50 bp duplex and 50mer ss-oligonucleotide were loaded as markers (C1 and C2 respectively). The nucleotides as indicated were present in a concentration of 2 mM. (C) Strand-annealing activity of RECQ5α and RECQ5β in the presence of the increasing concentration of RPA. The complementary 50-mer oligonucleotiddes (0.5 nM) were incubated with or without RPA for 5 min at room temperature. The RECQ5 proteins were added to a final concentration of 40 nM and incubation was continued at 37° C for 20 min. Reaction products were analyzed as in (A) and quantified using Image Quant software. The results are the average of three independent experiments. (D) FRET-based ssDNA-annealing assay. The annealing was measured by monitoring the decrease in fluorescence at 520 nm caused by FRET between fluorescein and

Figure 3 ATP

 3 Figure 3 ATP-binding affinity and ATPase activity of RECQ5 proteins (A) Changes in fluorescence intensity at 440 nm as 0.5 µM RECQ5α and RECQ5β, respectively, were titrated with increasing concentration of mantATP. Solid lines represent the best fit of the data to Equation 1. Apparent K d values are summarized in Table 1. (B) ATPase activities of wild-type and mutant RECQ5 proteins. The initial rate of ATP hydrolysis by RECQ5 proteins is plotted as function of the ATP concentration. Reactions contained 250 nM RECQ5 protein and 1.5 µM heated dsDNA and were carried out as described in Materials and Methods. The lines in the graph correspond to the best fits by Michaelis-Menten equation: V = V max [ATP]/(K M + [ATP]), where V is the initial reaction rate, [ATP] is the concentration of ATP, and K M is the Michaelis-Menten constant. Each value represents the mean of at least three independent measurements.

Figure 4

 4 Figure 4 Identification of the zinc-binding motif of RECQ5β helicase (A) Amino acid sequence alignment of the zinc-binding domains of RecQ family helicases. The multiple sequence alignment was performed using ClustalW software and refined manually. The conserved Cys and other residues are highlighted in red and blue, respectively. (B) Ribbon diagram showing the zinc-binding domain of the RECQ5β helicase as revealed by molecular modelling based on the crystal structure of E. coli RecQ. The zinc ion is in cyan. The positions of the four conserved cysteine residues are labelled as C1 to C4. The conserved arginine, aspartic acid and aromatic residues, involved in three very important hydrogen-bonds (dashed lines) that stabilize the conformation of the zinc-binding domain are shown in ball and stick

Figure 5 DNA

 5 Figure 5 DNA binding activities of wild-type and mutant RECQ5 proteins The anisotropy-based binding isotherms were obtained by varying protein concentration in the presence of 5 nM 3'-fluorescently labelled (A and C) ssDNA (BA) and (B and D) dsDNA (BA/BB). The data were fitted by Michaelis-Menten or

Figure 6 BiochemicalFig. 4 F374

 64 Figure 6 Model for the role of zinc-binding motif in the function of a RecQ helicaseIn the absence of the zinc-binding motif (left), the helicase domains bind ssDNA with low affinity. Although one part of ssDNA binds relatively tightly to the domain 1A, the other part of ssDNA may dissociate from domain 2A due to thermal fluctuation, providing an opportunity for domain 2A to capture a complementary ssDNA. In this situation, RECQ5α brings the two complementary strands into close proximity, promoting the hybridization of the complementary ssDNA. In this state, although ATP is bound tightly to domain 1A, the distance between the domains 1A and 2A is too long to form a functional ATPase site. In the presence of the zinc-binding motif (ZBM; right), the DNA substrate is tightly bound to the enzyme through an additional interaction between the zinc-binding motif and the duplex region of the substrate, which stabilizes ssDNA binding to the domains 1A and 2A. This tight DNA binding induces conformational changes in the protein that bring the two helicase subdomains into a closed form, allowing ATP hydrolysis, a prerequisite for the helicase function.

Fig. 6 Biochemical

 6 Fig. 5

Table 1 Summary of the measured parameters for RECQ5 isoforms and mutants

 1

	d	appa =218.17±7.8 nM and Hn= 2.68±0.18 for dsDNA and K d	appa =149.8 nM and Hn=2.76 for ssDNA

a Values corresponding to the fast rate constant and the slow rate constant, respectively. b DNA binding affinity is determined with K

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COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.