Simon Messner

Stephan Leitner

Christian Bommassar

Georg Golderer

Peter Gröbner

Ernst R Werner

Gabriele Werner-Felmayer
email: gabriele.werner-felmayer@i-med.ac.at

Physarum nitric oxide synthases: Genomic structures and enzymology of recombinant proteins

Keywords: Physarum polycephalum, nitric oxide synthase, tetrahydrobiopterin, arginine, flavin, heme nitric oxide, NOS, nitric oxide synthase, nNOS, neuronal NOS, iNOS, inducible NOS, e-NOS, endothelial NOS, TB, Terrific Broth, IPTG, isopropyl thiogalactoside

Physarum nitric oxide synthases

SYNOPSIS.

Physarum polycephalum expresses two closely related, calcium-independent nitric oxide synthases. In previous work, we showed that both nitric oxide synthases are induced during starvation and apparently play a functional role for sporulation. Here, we characterized the genomic structures of both Physarum nitric oxide synthases, expressed both enzymes recombinantly in bacteria, and characterized their biochemical properties. Whereas the overall genomic organization of Physarum nitric oxide synthase genes is comparable to various animal nitric oxide synthases, none of the exon-intron boundaries are conserved. Recombinant expression of clones with various N-termini identified N-terminal amino acids essential for enzyme activity, but not required for heme binding or dimerization, and suggests the usage of non-AUG start codons for Physarum nitric oxide synthases. Biochemical characterization of the two Physarum isoenzymes revealed different affinities for L-arginine, flavine mononucleotide and tetrahydrobiopterin.

INTRODUCTION

Nitric oxide (NO), a signalling and cytotoxic/cytoprotective agent, plays a key role in regulating the nervous, immune and cardiovascular systems of animals. It is synthesized by nitric oxide synthase (NOS, E.C. 1.14.13.39) which occurs as 3 isoforms and genes, i.e. NOS1, NOS2 and NOS3, also termed neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS), respectively. In addition, a number of tissue-and development-specific splice variants encoding variant NOS proteins have been identified [START_REF] Forstermann | Expressional control of the 'constitutive' isoforms of nitric oxide synthase (NOS I and NOS III)[END_REF][START_REF] Eissa | Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization[END_REF][START_REF] Tiscornia | Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro-and antiapoptotic culture conditions[END_REF][START_REF] Bros | Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1-specific promoter sequences[END_REF]. All three NOS isoenzymes second cloning site of the pET-Duet1 vector using SacI/NotI restriction digestion. This cDNA started at the putative start methionine [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] and also contained an N-terminal His-tag. Various N-termini of Physarum NOS form A were generated by introduction of an additional SalI site at the desired position (Quikchange II kit, Stratagene), cutting by SalI and re-ligating. Mutations were introduced by site-directed mutagenesis (Quikchange II kit). For elongating the N-terminus of Physarum NOS form B, 155 bp of the genomic sequence 5' to the putative start ATG were introduced using the Infusion cloning kit (Clontech). This region was confirmed to be identical to the cDNA 5'-region generated by RACE-PCR (see below). This clone was further modified introducing an additional SacI restriction site at the desired position (Quikchange II kit), cutting by SacI and re-ligating, thus leading to recombinant proteins with various N-termini. In addition, we introduced an N-terminal StrepTag [START_REF] Schmidt | The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins[END_REF] using the Quikchange II protocol into one form of physnosa (A1) and its almost inactive mutant (A1LD) to allow preparation of homogenous, active proteins.

5' Elongation of cDNA clones

In order to elongate 5' sequences of the previously published Physarum NOS form A and form B cDNA clones [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF], the Smart RACE cDNA amplification kit (Clontech) was used.

Bacterial overexpression of Physarum NOSs and preparation of homogenates

For overexpression, pET-Duet1 expression plasmids (see above) grown in NovaBlue (Novagen) cells and purified by the S.N.A.P. midi prep plasmid preparation kit (Invitrogen) were transformed into TUNER DE3 cells (Novagen). Bacterial starter cultures (5 ml) from single colonies were grown in Luria Bertani Broth (LB, Difco) with ampicillin (50 μg/ml) to an OD 600 of 0.8 at 37 °C and 220 rpm, then pelleted and diluted in 15 ml of fresh TB without ampicillin but supplemented with 1.5 % (v/v) ethanol in order to increase recovery of soluble protein by induction of a heat shock response [START_REF] Barroso | Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms[END_REF]. Aliquots for determining NOS activity at time zero were taken and cultures were supplemented with 450 μM 5-aminolevulinic acid (Sigma A-7793) and 3 μM riboflavin (Sigma R-4500) in order to provide sufficient heme and flavins and 50 μM isopropyl thiogalactoside (IPTG). Bacteria were grown for further 24 h at 20 °C at 220 rpm in the dark for NOS expression. For purification, this protocol was upscaled to 30 ml starter cultures and 400 ml expression cultures. TB (terrific broth) was used instead of LB for expression cultures. Bacteria from these cultures were resuspended in 40 ml buffer A (50 mM Tris/HCl, pH 8.0, containing 10 % (v/v) glycerol, 25 μM FAD, 25 μM FMN, 50 μM L-arginine, 5 μM tetrahydrobiopterin (6R-5,6,7,8-tetrahydro-L-biopterin, Schircks Laboratories, Jona, Switzerland), 5 mM dithio-erythritol (DTE), 1 mM phenylmethyl-sulphonylfluoride (PMSF), 0.2 mg/ml lysozyme). After incubation for 20 min at 25 °C at 200 rpm, cells were homogenized using a French press (20.000 psi, 2 times), and mixed with 110 ml buffer B (50 mM Tris/HCl, pH 8.0, containing 10 % (v/v) glycerol, 100 μM L-arginine, 10 μM tetrahydrobiopterin, 5 mM dithio-erythritol (DTE)).

Purification of recombinant Physarum NOSs

All steps were carried out at 4 ºC and all elution buffers contained 100 µM L-arginine and 10 µM tetrahydrobiopterin. Protein was precipitated using 35.6 g ammonium sulphate (45% of saturation) and stirring for 1 h followed by centrifugation at 48.000 x g for 20 min. The pellet was then resuspended in 20 ml of buffer B by stirring for 1 h and undissolved material was collected by centrifugation at 48.000 x g for 20 min. The supernatant was dialysed for 2 times 2 h versus 1 L of buffer C (buffer B without flavins) using a 50 K membrane (Pierce). The dialysate was adjusted to 0.15 M NaCl and then incubated with 2', 5' ADP-sepharose 4B (Amersham Pharmacia) for 1 h using a rocking mixer at a ratio of 1 part dialysate-protein to 2 parts ADP-sepharose (w/w). After washing 2 times with 10 ml buffer B containing 0.15 M NaCl and 1 time with 10 ml buffer B, protein was eluted from ADP-sepharose with 1 ml A c c e p t e d M a n u s c r i p t buffer B containing 10 mM NADPH. Protein determination at various steps was done using the Bradford Protein Assay reagent from Bio-Rad. To obtain homogenous active protein, the dimeric fractions of NOS were then collected by gel filtration on Superose12 10/300 GL (GE Healthcare), and were finally purified using the StrepTag affinity protocol [START_REF] Schmidt | The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins[END_REF].

Native Physarum NOS

Native Physarum NOS was purified from microplasmodia as described [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF].

NOS assay

Radiometric citrulline NOS assay was carried out as described before [START_REF] Werner-Felmayer | Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum[END_REF] using buffer B (see above) supplemented with 2 mM NADPH as assay buffer. Briefly, bacterial homogenates (bacteria from 2 ml of culture were diluted in 700 μl of buffer A, incubated for 10 min at 25 °C and 1000 rpm in an Eppendorf thermomixer and finally mixed for 10 sec with an Ultra-Turrax) or partially purified NOS (5-20 μl/assay corresponding to 10 -15 μg of protein in the case of bacterial homogenates or ~1 μg of protein when using purified enzyme) were incubated in assay buffer containing 40000 cpm of HPLC-purified 3 H-L-arginine (Amersham) in a final volume of 200 μl for 10 min at 25 °C on an Eppendorf thermomixer. The reaction was then stopped, separated over Dowex 50 W columns and the flow-through was subjected to scintillation counting. In some assays, concentrations of cofactors or substrate were varied. IC 50 , EC 50 and K M values were calculated with the SigmaPlot 9.0 software (Systat Software Inc.).

For comparison of NO formation with NADPH consumption, a kinetic microtiter-plate assay based on measurement of NO formation by binding to myoglobin (monitored by UV absorption at 405 nm) combined with measurement of NADPH consumption (monitored at 340 nm) was performed at 25ºC as described [START_REF] Gross | Microtiter plate assay for determining kinetics of nitric oxide synthesis[END_REF], using a PowerWaveX thermostatted kinetic microplate reader (Bio-Tek Instruments,,Winooski, VT, U.S.A.).

Gel electrophoresis

Proteins were separated over SDS polyacrylamide gels. Gels were stained by Coomassie Blue or silver stain according to standard procedures and scanned using an ImageScanner (Amersham Biosciences). For quantification, gels were stained with Deep Purple Total Protein Stain (Amersham Biosciences) and fluorescence was scanned by a Typhoon 9410 scanner (Amersham Biosciences) at 532 nm and evaluated using the ImageQuant software (Amersham Biosciences).

Determination of heme content by HPLC

Heme content of pure forms A1 and A1LD was checked by HPLC according to Bonkovsky et al. [START_REF] Bonkovsky | High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials[END_REF] by reversed phase HPLC separation of porphyrins and UV detection at 405 nm. Suitability of the application of this method for the analysis of protein-associated heme was confirmed with equine myoglobin (Serva, Heidelberg, Germany) yielding 0.9 ± 0.1 heme per myoglobin.

RESULTS

Expression of Physarum NOS forms A and B mRNA

We first wanted to clarify whether the two highly related Physarum NOS isoforms [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] are the products of two genes or of two alleles of the same gene like it was shown for the Physarum hapP gene [START_REF] Lepine | The two alleles of the hapP gene in Physarum polycephalum code for different proteins[END_REF]. For this purpose, we used the haploid Physarum strain LU352 and checked for expression of Physarum NOS forms A and B mRNA. Both mRNAs were expressed in LU352 macroplasmodia starved for 5 days in a way comparable to the diploid M 3 b strain which we used in our initial studies [START_REF] Werner-Felmayer | Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum[END_REF][START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. Moreover, LU352 amoebae also expressed both mRNAs (Fig. 1).

Genomic structures of both Physarum NOSs

We then studied the genomic structures of both Physarum NOSs. As can be seen from Fig. 2A, 25 exons could be identified for Physarum NOS form B. Form A has two additional 5' exons as was determined extending the cDNA into the 5' direction, a strategy which was not successful for the form B cDNA where only a shorter 5' extension could be achieved starting within an exon (Fig. 2A). On the other hand, the exact number of exons for NOS form A could not be determined since some internal genomic regions were inaccessible to sequencing (Fig. 2A). However, since all the 21 exon/intron boundaries that could be determined were conserved between the two Physarum NOSs, it is conceivable that also the lacking 5 boundaries are conserved. We therefore concluded that the two genes consist of 27 exons. For comparison, the gene structures of iNOS from human, chicken and trout are similarly organized into 27, 28, and 27 exons, respectively (Fig. 2A). However, none of the exon/intron boundaries are conserved between Physarum and the animal iNOSs (Fig. 2A). Moreover, the size of introns in the Physarum genes is considerably smaller as compared to animal NOSs, particularly the human iNOS gene (Fig. 2B). As in other animal iNOSs, the reading frame of the Physarum NOSs starts in exon 3 (Fig. 2A). The current version of the two gene structures was submitted to GenBank (DQ835529, DQ845107 and DQ835525 for NOS form A and DQ835527 for NOS form B). The extended cDNA sequences were also submitted: DQ835526 for NOS form A and DQ825528 for NOS form B. The final gene structures will become available soon through the ongoing Physarum genome project (http://www.genome.gov/12511858 and http://genome.wustl.edu/genome.cgi?GENOME =Physarum% 20polycephalum).

Recombinant overexpression of Physarum NOSs in Escherichia coli

Our next goal was to set up efficient recombinant overexpression of Physarum NOSs in E. coli. In previous work, we could functionally express Physarum NOS form A in a baculovirus system [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] but expression levels were low and did not allow purification of recombinant protein. It is known that high-level expression of mouse iNOS requires coexpression of calmodulin [START_REF] Wu | High-level expression of mouse inducible nitric oxide synthase in Escherichia coli requires coexpression with calmodulin[END_REF]. We therefore cloned Physarum calmodulin and coexpressed it together with Physarum NOS form A and B, respectively, using the pET-Duet1 vector. In contrast to the original Physarum NOS form A cDNA library clone (AF145041) which still contained 104 additional amino acids 5' to the putative start methionine [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] and a variant A1, which contained only 29 amino acids 5' to the putative start, the clone starting at the predicted methionine (A2) had no enzyme activity (Fig. 3A), although it expressed the same amount of protein (Fig. 3D). Elimination of vector-derived sequences including the His-Tag resulted in disappearance of both, protein and activity, presumably due to proteolytic degradation (not shown). Systematically shortening the N-terminus 5' to the assumed start methionine then revealed that 13 amino acids were sufficient to achieve a clone with enzyme activity (variant A8) which dropped dramatically when one amino acid (leucine) was cut off (variant A9) and became undetectable when deleting a further amino acid (variant A3) (Fig. 3A). Exchanging this leucine residue in A1 for aspartic acid or for glycine (variants A1LD and A1LG) destroyed enzyme activity almost completely (Fig. 3A), while it did not affect the amount of overexpressed protein (Fig. 3B), and did not affect the heme content of homogenously purifed proteins (see below), nor the monomer-dimer ratio on gel filtration (not shown). Expressing Physarum NOS form B using the predicted start methionine (clone B) also resulted in an inactive protein while addition of 50 amino acids 5' to this methionine (clone B+) yielded active protein. Varying this N-terminus showed that only 4 additional amino acids were sufficient for full activity of Physarum NOS form B (Fig. 3A). Like in form A, removal of a hydrophobic amino acid (leucine for truncation from B6 to B7, isoleucine for truncation from B9 to B8) leads to the drop in activity, although the amount of NOS protein in inactive clones is comparable to that of active clones (Fig. 3E). These results clearly indicated that the Nterminus of both Physarum NOSs as predicted before [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] may be too short. In line with this, comparison of the proteins expressed from the A and B clones and their variants with native Physarum NOS showed that the molecular masses of several of these proteins were smaller than the native protein which is a mixture of both isoforms [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] (Fig. 3C).

Checking the elongated Physarum NOS cDNAs showed that form A cDNA (DQ835526) contained a stop codon 516 bp up-stream of the predicted start ATG but no additional methionine codon whereas the form B cDNA (DQ835528) had no stop codon over the whole cloned 740 bp region 5' to the putative start codon but encoded for an additional methionine 69 amino acids up-stream of the predicted start [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] (Fig. 4A). Starting with this methionine, the predicted form B protein (Fig. 4B) is still smaller than the native Physarum NOS (Fig. 3C). Searching the cDNA sequence up-stream of the putative start codon showed that both Physarum NOSs have several putative non-AUG start codons [START_REF] Takahashi | Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2)[END_REF][START_REF] Kozak | Regulation of translation via mRNA structure in prokaryotes and eukaryotes[END_REF]. An isoleucine conserved in both Physarum NOSs is found 88 (form A) or 90 (form B) amino acids upstream (Fig. 4A). However, the Kozak sequence for these codons is less optimal [START_REF] Bailey | Growth and Development in Relation to the Cell-Cycle in Physarum-Polycephalum[END_REF] than that of other nearby residues (Fig. 4B): for Physarum NOS form A cDNA this is the isoleucine codon (ATT) at amino acid position -97; for Physarum NOS form B, an alternative start codon (ATT) with a nearly optimal context is encoding for isoleucine in position -108. These sites yield proteins of nearly identical molecular mass (Fig. 4B) which are comparable to that of native NOS (Fig. 3C).

Enzymatic properties of recombinant Physarum NOSs

We then characterized enzymatic properties of recombinant Physarum NOSs. For this purpose, we used proteins expressed from the A1 and B+ clone partially purified by ammonium sulphate precipitation and subsequent ADP-sepharose affinity chromatography. This procedure yielded NOS with about 80 % purity, as determined by scanning bands stained with a fluorescence dye (see Experimental Procedures, data not shown, compare Fig. 5A, last lane). The specific activity was 244 ± 47 nmol.mg -1 .min -1 (form A, mean of 3 purifications ± SD) and 253 ± 47 nmol.mg -1 .min -1 (form B, mean of 4 purifications ± SD). Fig. 5 shows a typical purification of A1 recombinant protein. While both Physarum NOSs were comparably sensitive to NOS inhibitors and similarly dependent on NADPH and FAD, and had similar dimer-monomer ratios of about 1:1 on gel filtration (Figure 6 A, B), the K M for L-arginine and the EC 50 for FMN and tetrahydrobiopterin was clearly higher for NOS form A than for the form B protein (Table 1). A combined NO formation (determined by binding to myoglobin) and NADPH consumption assay [START_REF] Gross | Microtiter plate assay for determining kinetics of nitric oxide synthesis[END_REF] showed partial decoupling of the enzyme preparations, with 5.1 ± 1.6 NADPH consumed in the formation of 1 NO molecule by Physarum NOS A and 5.9 ± 1.5 NADPH consumed per NO in Physarum NOS B (mean ± SD of three determinations).

Heme content of homogenous Physarum NOSs A1 and A1LD

To check whether or not the mutation of a residue in the N-terminus that led to a pronounced drop in activity also affected heme binding and hence heme content of the active enzyme, we introduced into forms A1 and A1 LD (see Fig. 3A) a StrepTag, and further purified the tagged proteins by additional gel filtration, collection of the dimeric fractions and final purification using the StrepTag affinity column. This yielded a homogenous protein (> 95 % SDS gel silver stained, Fig. 6C) free of E.coli impurities (minor impurities also contain the StrepTag as checked by Western blotting). Pure A1 (3 preparations) had a specific activity of 585 ± 144 nmol.mg -1 .min -1 , and a heme content of 0.41 ± 0.14 heme per subunit, whereas pure A1LD (2 preparations) displayed less than a tenth of the activity (51 ± 10 nmol.mg -1 .min - 1 ), but had a heme content (0.44 ± 0.09 per subunit) comparable to A1 (Table 2).

DISCUSSION

Physarum polycephalum, a member of the class/superclass Myxogastridae (also termed myxomycetes or true slime molds), is the only so far identified non-animal species known to express NOS with the characteristics of mammalian iNOS [START_REF] Werner-Felmayer | Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum[END_REF]. Previously, we showed that this enzyme is induced by starvation of macroplasmodia and plays a crucial role in sporulation [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. Cloning experiments indicated that Physarum contains two highly related calcium-independent NOSs (forms A and B, 82 % amino acid identity) that are expressed in parallel throughout various developmental stages and have a similar molecular mass, as was not only deduced from the cDNA sequences but was underlined by the fact that purified native Physarum NOS was a mixture of both proteins that were not separated by SDS gel electrophoresis [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. While parallel expression of calcium-dependent and calciumindependent NOS isoforms within a certain cell type is common in animal species, the parallel occurrence of two versions of calcium-independent NOS was never reported, at least to our knowledge. The previously used Physarum strain was the diploid M 3 b isolate. Thus, it was possible that the two different cDNAs and proteins could stem from two alleles of the same gene, as it was described for the plasmodium-specific hapP mRNA which encodes proteins of unknown function differing by 9.6 % in their predicted amino acid sequence [START_REF] Lepine | The two alleles of the hapP gene in Physarum polycephalum code for different proteins[END_REF]. We therefore checked for expression of Physarum NOS form A and form B in the haploid Physarum strain LU352 [START_REF] Barroso | Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms[END_REF], the strain now being used for sequencing the Physarum genome (http://www.genome.gov/12511858 and http://genome.wustl.edu/genome.cgi?GENOME =Physarum% 20polycephalum). Quantifying Physarum NOS form A and form B expression in starved macroplasmodia of both strains showed comparable mRNA levels. Moreover, both mRNAs were also expressed in LU352 amoebae. Macroplasmodia of diploid strains develop after fusion of genetically different amoebae but in the apogamic haploid strains like LU352 no such fusion is required [START_REF] Dee | Growth, Development and Genetic-Characteristics of Physarum-Polycephalum Amebas Able to Grow in Liquid, Axenic Medium[END_REF][START_REF] Bailey | Growth and Development in Relation to the Cell-Cycle in Physarum-Polycephalum[END_REF]. Therefore, expression of both NOS mRNAs in LU352 amoebae underlined the assumption that the two Physarum NOS forms stem from two genes rather than from two alleles of the same gene.

Studying the organization of these two genes revealed a high similarity of the overall structure of the Physarum NOS genes to those of NOS genes from higher animals including mammals. Intron size of Physarum NOSs is smaller than that of animal NOSs which is in agreement with the smaller genome size of Physarum (300 Mb, personal communication of Gérard Pierron, Villejuif, France) as compared to human (3038 Mb) or chicken (1200 Mb, http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi) [START_REF] Charlesworth | Genome size: does bigger mean worse?[END_REF]. None of the intron positions is conserved between Physarum and animal NOS genes which is in line with the idea that introns are transposable elements that are aquired or also deleted during evolution [reviewed in 32]. Transposition of introns occurs very infrequently during evolution, for example most orthologous genes between mouse and human, species that diverged at least 50 million years ago, have the same intron positions [START_REF] Fedorova | Introns in gene evolution[END_REF]. This gives some idea of the evolutionary distance between Physarum and higher animals and their early divergence from a common ancestor (see below) where already some ancestral NOS had evolved. Once the final Physarum genome sequence will be available, these speculations can be studied in more detail and it will also be possible to clarify whether the two Physarum NOS genes stem from single gene duplication or other evolutionary events. Some further interesting aspects with regard to evolution become evident here. According to molecular phylogenetic data, Physarum is most closely related to the cellular slime molds, the Dictyostelidae [START_REF] Baldauf | Origin and evolution of the slime molds (Mycetozoa)[END_REF][START_REF] Baldauf | A kingdom-level phylogeny of eukaryotes based on combined protein data[END_REF]. Together with other amoebae and slime mold classes they are grouped as Amoebozoa, one of eight major groups composing the "crown" of the phylogenetic tree of eukaryotes [START_REF] Baldauf | Origin and evolution of the slime molds (Mycetozoa)[END_REF][START_REF] Baldauf | The deep roots of eukaryotes[END_REF], thus indicating that these organisms cannot be considered as "low" stages on the path to higher animals and fungi but have developed independently from a more ancient, not yet defined root [START_REF] Baldauf | The deep roots of eukaryotes[END_REF]. However, a more refined analysis of evolutionary relationships between Physarum and Dictyostelium, another model organism for cell biology, has to await the final Physarum genome. In any case, our data on NOS as well as previous observations concerning pteridine biosynthesis (see below) underline the diversity of Physarum and Dictyostelium despite the striking similarities of their life cycle, the occurrence of mobile and stationary developmental stages, their habitat and their way of feeding as well as the fact that their protein sequences usually group on a common branch in phylogenetic analysis. First of all, Dictyostelium discoideum does not contain any gene similar to full-length NOS (as checked by Blast-P search of the Dictyostelium genome with the Physarum NOSs, not shown), while Physarum contains two genes with exon numbers comparable to those of higher animals (see Fig. 2) and an even intron distribution as it is characteristic for higher eukaryotes [START_REF] Mourier | Eukaryotic intron loss[END_REF]. Secondly, Dictyostelium does not produce tetrahydrobiopterin like Physarum [START_REF] Werner-Felmayer | Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum[END_REF] but its steroisomer tetrahydrodictyopterin [START_REF] Klein | Dictyopterin, 6-(D-threo-1,2dihydroxypropyl)-pterin, a new natural isomer of L-biopterin. Isolation from vegetative cells of Dictyostelium discoideum and identification[END_REF], a pterin that cannot serve as a cofactor for mammalian NOS [START_REF] Werner | Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase[END_REF] or Physarum NOS (E.R. Werner, G. Golderer, P. Gröbner and G. Werner-Felmayer, unpublished data). Moreover, the genomic structures of GTP cyclohydrolase I, the first enzyme of tetrahydrobiopterin biosynthesis, differ significantly between the two species [START_REF] Golderer | GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing[END_REF]: in accordance with its comparatively small genome of 34 Mb (http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi), Dictyostelium has only one 109 bp intron [START_REF] Witter | Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum[END_REF], whose location is conserved in Physarum, whereas the Physarum gene consists of 7 exons and thus resembles animal GTP cyclohydrolase I genes with some intron positions conserved in Drosophila or human. In addition, alternative splicing of GTP cyclohydrolase I occurs in Physarum at a similar position to human [START_REF] Golderer | GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing[END_REF]. Taken together, these findings suggest that Physarum may be more closely related to animals than Dictyostelium with respect of tetrahydrobiopterin and NO synthesis, and it underlines the value of Physarum as a model system for studying certain aspects of animal cell biology. In line with our observations, the recent analysis of Physarum expressed sequence tags [START_REF] Gloeckner | A first glimpse at the transcriptome of Physarum polycephalum[END_REF] has revealed 895 genes of Physarum with similarities in other databases but not in the Dictyostelium protein database. In addition, about 9 % of all detected genes are alternatively spliced in Physarum, whereas only about 0.2 % of genes are alternatively spliced in Dictyostelium [START_REF] Gloeckner | A first glimpse at the transcriptome of Physarum polycephalum[END_REF].

In the course of setting up recombinant expression of both Physarum NOSs in bacteria, we found that the previously predicted reading frame [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] was too short at its N-terminus. We concluded this not only from lack of activity of His-tagged expression constructs, but also from the smaller size of the recombinant proteins as compared to the native NOS purified from Physarum microplasmodia (Fig. 3C). Addition of only a few amino acids (13 for NOS form A and 4 for NOS form B) to the N-terminus as deduced from the cDNAs led to expression of functional proteins. Producing 5'-extended cDNA sequences for both NOSs and checking for other possible translation starts revealed that only NOS form B had an up-stream methionine within reasonable distance, whereas for NOS form A we found a stop codon and no additional AUG-codons between this stop and the previously predicted start. Thus, at least NOS form A appears to use a non-AUG start codon [START_REF] Kozak | Regulation of translation via mRNA structure in prokaryotes and eukaryotes[END_REF] and this may also be the case for NOS form B. The molecular masses of the predicted NOSs starting from these alternative start codons (both encoding for isoleucine) compare much better to native NOS than those of the proteins starting with the first encoded AUG codon. It should be noted that none of the possible AUG codons have an optimal context whereas the predicted alternative non-AUG codons, particularly of NOS form B, fit much better to this rule [START_REF] Kozak | Regulation of translation via mRNA structure in prokaryotes and eukaryotes[END_REF]. Future studies will clarify the exact start of Physarum NOS proteins but at least NOS form A and presumably also form B appear to belong to the growing group of mRNAs with non-AUG translation A c c e p t e d M a n u s c r i p t initiation [START_REF] Kozak | Regulation of translation via mRNA structure in prokaryotes and eukaryotes[END_REF]. Sometimes, like was shown for the mammalian translational regulator NAT1 [START_REF] Takahashi | Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2)[END_REF], usage of a non-AUG translation initiation site is even conserved evolutionarily. However, there is no report that any of the animal NOS mRNAs starts translation at a non-AUG codon.

Although the two isoforms of Physarum NOS are highly similar, we found distinct differences in the enzymology of the recombinat proteins expressed in E. coli. Physarum NOS form B has a significantly higher affinity towards L-arginine, tetrahydrobiopterin and FMN than Physarum NOS form A. The purified recombinant proteins of form A and B contained about the same amount of dimer and monomer, and were partly decoupled in that they consumed more than 5 NAPDH per NO formed rather than 1.5 NADPH per NO as the fully coupled mammalian enzyme [START_REF] Gorren | Nitric-oxide synthase: a cytochrome P450 family foster child[END_REF].

We were surprised to find the requirement of N-terminal amino acid residues upstream of the first AUG codon since this is a region with low homology to other NOSs, and a region which iNOS oxygenase domains apparently do not require for function [START_REF] Ghosh | Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction[END_REF]. In iNOS oxygenase domain crystals, this region is disordered [START_REF] Crane | Structure of nitric oxide synthase oxygenase dimer with pterin and substrate[END_REF] so that no structural information of this region is available to date. After we had determined the minimal amount of residues required to yield a functional recombinant protein, we mutated the amino acid upon the truncation of which the activity was lost, a leucine, in a larger construct to investigate whether we simply required a certain length of the protein, or whether specific residues were needed. Activity was lost upon mutation of this leucine to aspartate or glycine, without affecting the monomer-dimer ratio and the heme content of homogenously purified proteins. We assume that specific residues in this region are possibly required for interactions between oxygenase and reductase domains in the dimer of the enzyme, for which hydrophobic forces might be crucial.

Table 1. Enzymatic properties of recombinant

concentration (µM)

Physarum A c c e p t e d M a n u s c r i p t [START_REF] Crane | Structure of nitric oxide synthase oxygenase dimer with pterin and substrate[END_REF][START_REF] Bredt | Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase[END_REF][START_REF] Raman | Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center[END_REF][START_REF] Fischmann | Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation[END_REF][START_REF] Garcia-Cardena | Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo[END_REF]. For Physarum NOS form A, some internal exon/intron boundaries could not be determined by sequencing but were deduced from form B. This region is shown in white and the 5 deduced exon/exon boundaries are indicated by a thin line (bar 4). Also, exon 18 could not be fully sequenced. For Physarum NOS form B, the 5' region could only be partially extended and starts within an exon (bar 5, indicated by interrupting the margin at the 5' end) which was assigned number 3 as deduced from homology to NOS form A. B, Intron sizes (bp) (GenBank accessions for genomic and cDNA sequences are given under A) from iNOS genes of human (huminos), chicken (galinos), trout (oncinos) and the two Physarum NOSs (physnosa; physnosb). ns, not sequenced; -, not identified. The previously predicted start of translation is underlined [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. All overexpressed proteins start with a His-tag sequence (MGSSHHHHHHSQDPNSSSAR LQVD), indicated by 3 dots. Clone A has an additional GIDKLDIEFRSKG sequence from the Bluescript SK -vector inserted between the His-tag and the Physarum NOS sequence. aa, amino acids. B, Overexpression of A1 and its A1LD mutant. Raw homogenates (15 μg) harvested at times 0 and 24 h after induction were separated over 6 % SDS polyacrylamide gels and stained with Coomassie Blue. For the induced cultures of A1 and A1LD, samples from two different expressions are shown. C, Comparison of overexpressed Physarum NOS from clones A, A1, and B+ (3 μg protein of raw homogenates per lane) with native NOS purified from Physarum microplasmodia (0.3 μg/lane) [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. Bands were detected by silver staining. Sequencing demonstrated that the second band in native Physarum NOS is a proteolytic fragment of the first one, and that both bands contained a mixture of Physarum NOS form A and Physarum NOS form B [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. D, Silver stained gel of various A clones (raw homogenates, 3 μg of protein/lane). E, Silver stained gel of various B clones (raw homogenates, 3 μg/lane). Molecular masses (kDa) of several recombinant proteins were calculated for proteins containing the His-tag and are given below the lanes of panels C-E. [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] is framed. An additional methionine in form B is marked by a grey arrow, putative alternative starts of the reading frames are indicated by black arrows. Italic numbers refer to amino acid positions related to the previously predicted reading frames [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF], normal numbers refer to amino acids deduced from the 5' extended cDNA clones (DQ835526 and DQ835528). The asterisk denotes the stop found in the Physarum NOS form A cDNA. B, Molecular mass for Physarum NOSs with various starts and the Kozak sequences of their putative start codons. Putative start codons are written in small letters whereas the context for translation is given in large letters. The nucleotide positions as well as the amino acid positions given in brackets refer to GenBank sequences DQ835526 and DQ835528. Amino acid positions not in brackets refer to the previously predicted reading frame [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF] starting with methionine 1. The asterisk indicates previously published data [START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. . 20 μg of total protein were loaded per lane; homogenate, raw bacterial homogenate; supernatant, supernatant after loading the 2',5' ADP sepharose 4B column; wash, first wash of 2',5' ADP sepharose 4B with buffer B; eluate, protein eluted from 2',5' ADP sepharose 4B with buffer B containing 10 mM NADPH. The ammonium-sulphate precipitation step is not shown. B, Activities, yield and purification factor of Physarum NOS form A clone A1 protein purified from 400 ml bacterial culture (homogenate) by ammonium-sulphate precipitation (AS-precipitation) and 2',5' ADP sepharose 4B affinity chromatography (2'5'-ADP).

Biochemical

  Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192

Biochemical

  Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

  Biochemical Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192

  Physarum NOSs. Partially purified recombinant Physarum NOS form A (clone A1) and form B (clone B+) with an activity of about 250 nmol.mg -1 .min -1 each (see text for data) was used for measuring substrate, cofactor and inhibitor effects. H 4 -bip, tetrahydrobiopterin; L-NIL, L-N6(1-iminoethyl)-lysine; L-NNA, N ω -nitro-L-arginine; L-NMMA, N ω -methyl-L-arginine. Values are means of 3 to 5 independent experiments ± SD. Values in bold are significantly different (Student's t-test) between NOS form A and form B: K M L-arginine, P < 0.001; EC 50 tetrahydrobiopterin, P < 0.001; EC 50 FMN, P < 0.02.

Figure 1 .

 1 Figure 1. Quantification of Physarum NOS form A and form B mRNA levels. Total RNA was isolated from starved macroplasmodia of the diploid M 3 b and the haploid LU352 strain as well as from LU352 amoebae and the two NOS forms were quantified by Taqman technology in relation to 19 S RNA. The mean of 3 independent experiments ± SD is shown. The dashed line indicates the detection limit of the method. 1: M 3 b macroplasmodia, Physarum NOS form A; 2: M 3 b macroplasmodia, Physarum NOS form B; 3: LU352 macroplasmodia, Physarum NOS form A; 4: LU352 macroplasmodia, Physarum NOS form B; 5: LU 352 amoebae, Physarum NOS form A; 6: LU352 amoebae, Physarum NOS form B.

Figure 2 .

 2 Figure 2. Genomic organization of Physarum NOSs. A, Exons in cDNAs of human iNOS (bar 1, 27 exons; GenBank accession for genomic sequence: NT_010799.14; cDNA: NM_000625), chicken iNOS (bar 2, 28 exons; genomic: NW_060634.1|Gga19_WGA443_1; cDNA: NM_204961), trout iNOS (bar 3, 27 exons; genomic: AJ295231; cDNA: AJ295230), Physarum NOS form A (bar 4, 27 exons; genomic: DQ835529 (5' region, 6 exons), DQ845107 (internal region, 5 exons), DQ835525 (3' region, 9 exons); cDNA: DQ835526) and form B (bar 5, 27 exons; genomic: DQ835527; cDNA: DQ835528). Lines below each bar indicate the translated region of the cDNA. Positions of various binding sites are marked as published before[START_REF] Crane | Structure of nitric oxide synthase oxygenase dimer with pterin and substrate[END_REF][START_REF] Bredt | Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase[END_REF][START_REF] Raman | Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center[END_REF][START_REF] Fischmann | Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation[END_REF][START_REF] Garcia-Cardena | Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo[END_REF]. For Physarum NOS form A, some internal exon/intron boundaries could not be determined by sequencing but were deduced from form B. This region is shown in white and the 5 deduced exon/exon boundaries are indicated by a thin line (bar 4). Also, exon 18 could not be fully sequenced. For Physarum NOS form B, the 5' region could only be partially extended and starts within an exon (bar 5, indicated by interrupting the margin at the 5' end) which was assigned number 3 as deduced from homology to NOS form A. B, Intron sizes (bp) (GenBank accessions for genomic and cDNA sequences are given under A) from iNOS genes of human (huminos), chicken (galinos), trout (oncinos) and the two Physarum NOSs (physnosa; physnosb). ns, not sequenced; -, not identified.

Figure 3 .

 3 Figure 3. Overexpression of recombinant Physarum NOSs. A, N-termini of various clones of Physarum NOS form A and form B and their activity in raw homogenates. Values are means of 5-10 independent experiments ± SD in brackets.The previously predicted start of translation is underlined[START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. All overexpressed proteins start with a His-tag sequence (MGSSHHHHHHSQDPNSSSAR LQVD), indicated by 3 dots. Clone A has an additional GIDKLDIEFRSKG sequence from the Bluescript SK -vector inserted between the His-tag and the Physarum NOS sequence. aa, amino acids. B, Overexpression of A1 and its A1LD mutant. Raw homogenates (15 μg) harvested at times 0 and 24 h after induction were separated over 6 % SDS polyacrylamide gels and stained with Coomassie Blue. For the induced cultures of A1 and A1LD, samples from two different expressions are shown. C, Comparison of overexpressed Physarum NOS from clones A, A1, and B+ (3 μg protein of raw homogenates per lane) with native NOS purified from Physarum microplasmodia (0.3 μg/lane)[START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. Bands were detected by silver staining. Sequencing demonstrated that the second band in native Physarum NOS is a proteolytic fragment of the first one, and that both bands contained a mixture of Physarum NOS form A and Physarum NOS form B[START_REF] Golderer | Nitric oxide synthase is induced in sporulation of Physarum polycephalum[END_REF]. D, Silver stained gel of various A clones (raw homogenates, 3 μg of protein/lane). E, Silver stained gel of various B clones (raw homogenates, 3 μg/lane). Molecular masses (kDa) of several recombinant proteins were calculated for proteins containing the His-tag and are given below the lanes of panels C-E.

Figure 4 .

 4 Figure 4. Putative translation start sites in Physarum NOSs. A, Amino acid sequence predicted from 5' extended cDNA clones of Physarum NOS form A (physnosa, GenBank DQ835526) and form B (physnosb, DQ835528). Conserved residues are shown in bold. The previously predicted start of the protein [18] is framed. An additional methionine in form B is

Figure 5 .

 5 Figure 5. Partial purification of recombinant Physarum NOS form A (clone A1). A, 6-17 % gradient, Coommassie-Blue-stained SDS polyacrylamide gel showing partial purification of recombinant Physarum NOS form A clone A1. 20 μg of total protein were loaded per lane; homogenate, raw bacterial homogenate; supernatant, supernatant after loading the 2',5' ADP sepharose 4B column; wash, first wash of 2',5' ADP sepharose 4B with buffer B; eluate, protein eluted from 2',5' ADP sepharose 4B with buffer B containing 10 mM NADPH. The ammonium-sulphate precipitation step is not shown. B, Activities, yield and purification factor of Physarum NOS form A clone A1 protein purified from 400 ml bacterial culture (homogenate) by ammonium-sulphate precipitation (AS-precipitation) and 2',5' ADP sepharose 4B affinity chromatography (2'5'-ADP).

Figure 6 .5

 6 Figure 6. Gel filtration of purified Physarum NOSs Physarum NOS isoforms (clones A1 and B+, respectively) carrying an N-terminal Strep tag were expressed together with Physarum calmodulin in E. coli using the pETDuet1 vector (see Materials and Methods for details). 200 µl of purified protein was then loaded onto a Superose 12 10/300 GL gel filtration column, eluted with buffer B containing 0.15 M NaCl. Fractions were collected and NOS activity was determined by the radiometric citrulline assay. The full line shows UV absorption at 280 nm, the dashed line shows NOS activity. Arrows indicate the elution volume of protein standards for gel filtration (thyreoglobulin, 690 kDa; ßamylase, 200 kDa; albumin, 67 kDa). A, Physarum NOS A, B, Physarum NOS B. C, Silverstained SDS gel showing the purity of Strep-tagged Physarum NOS A.

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  Messner et al., Figure 4 Biochemical Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Messner et al., Figure 5 Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

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Table 2 Original data of heme determination of pure Physarum NOS preparations.

 2 Physarum NOS clones A1 and A1LD containing an N-terminal Strep tag, were coexpressed with Physarum calmodulin in E.coli with pETDuet1 and purified by ammonium sulphate fractionation, ADP-sepharose affinity chromatography, gel filtration and Strep-tag affinity chromatography. Heme was determined in purified fractions by reversed phase HPLC with UV detection. NOS activity was determined by the radiometric citrulline assay. Protein was determined by the Bradford assay using bovine serum albumin as standard. MM (calculated molecular mass). See Materials and Methods section for method details.

	NOS A	Physarum NOS B

Batch Heme (µM) Protein (mg/ml) MM (kDa) Heme/ subunit NOS activity (nmol/mg.min)

  Biochemical Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192

	NOSA1	1	2.57	1.30	126	0.25	768
	NOSA1	2	7.47	2.32	126	0.41	417
	NOSA1	3	4.71	1.02	126	0.58	569
	NOSA1-LD	1	5.01	1.80	126	0.35	41
	NOSA1-LD	2	4.16	1.00	126	0.52	61

  physnosa ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ 0 physnosb YPIMPNTPHA APHDSPASVL PRSSPRTTTP SPPPIPIRPH PITPPWPKRE NALSSLPRDE 60 physnosa ~~~~~~~~TS KKKSSAQFPV LPTSFPILPG GESKALTSPR PSTSSNTTSS FPILPNSNAE 52 physnosb SAFHETSGIT RRKDTPSTPT HNSNFPILP. .STRAITHPP TPPADPTPAT QTTPPAQNLP 118 physnosa P.SKSAYPV. ..LPNGETSK GSARPAIPTL P...TSFPIL PS..ADSSHA PARTTPRDVA 103 physnosb PLNFSGLPAS DESASSETSG ITRRNHSPST PAHFSNYPIL PSSRASTPTT STTTTANPPM 178 physnosa PAHGTNYPIL PSASQELVPA APSHSHAAPN PAHAPHPVPA NSTGQCPFLN AFNNP..... 158 physnosb PSTAAHPPTN PTLATEI..S APAQN...PN PFHAPHPASS LSSGQCPFLN AFSTDTTHAH 233

	A
		*
					-97		-88
				-108		-90	-69
				1
	physnosa LPSHAHKGPL PIYMLTNN 176
	physnosb IPPHAHKGPL PIYMLTNN 251
				1
	B
	A	Met	1	(72)	844	1055*	118180,89*	AUCUACaugU
	A	Ile	-88 (84)	580	1143	127231,88	UUCCCCaucC
	A	Ile	-97 (75)	553	1152	128185,98	CCUGCCaucC
	B	Met	1 (247)	740	1046*	117566,52*	AUCUACaugC
	B	Met	-69 (178)	533	1115	124814,32	CCUCCCaugC
	B	Ile	-90 (157)	491	1136	126827,70	UAUCCGaucC
	B	Ile	-108 (139)	416	1154	128891,94	UCCGGCauuA

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Messner et al., Physarum nitric oxide synthases

Biochemical Journal Immediate Publication. Published on 01 Dec 2008 as manuscript BJ20080192 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080192 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

ACKNOWLEDGMENTS.

We thank Renate Kaus, Petra Loitzl and Nina Madl for excellent technical assistance. We also thank Wolfgang Marwan, University of Magdeburg, Germany, for providing the LU352 strain and Jonatha Gott, Case Western Reserve University, Cleveland, USA, for the Physarum genomic library. This work was supported by the Austrian Funds "Zur Förderung der wissenschaftlichen Forschung", projects P15538 (to G.G.) and P16059 (to G.W.-F.), and by the Tiroler Wissenschaftsfonds UNI-0404-245 (to G.W.-F.).