Carlos Rouco
email: carlos.rouco@uclm.es

Rafael Villafuerte
email: rafael.villafuerte@uclm.es

Francisca Castro
email: francisca.castro@uclm.es

Pablo Ferreras
email: pablo.ferreras@uclm.es

Responses of naïve and experienced European rabbits to predator odour

Keywords: Artificial warren, Behaviour, Oryctolagus cuniculus, Pellet counts, Predation risk

come

Introduction

Predation is a central feature of ecological communities. Most studies of predator-prey interactions have focused on the capture or consumption of prey by predators; however, predator effects on prey are not limited to predation because it is known that a prey can respond to predators by altering phenotypic traits to reduce the risk of mortality [START_REF] Preisser | Scared to death? The effects of intimidation and consumption in predator-prey interactions[END_REF]. Prey responses do not require the presence of predators, they can be induced by cues of their presence, for instance their scent. Many types of responses to predator scent have been investigated among prey species. Spatial avoidance and changes in foraging behaviour have been observed in rodents when a predator cue or scent is present [START_REF] Wolff | Response of gray-tailed voles to odours of a mustelid predator: a field test[END_REF][START_REF] Ylönen | Is the antipredatory response in behaviour reflected in stress measured in faecal corticosteroids in a small rodent?[END_REF].

Several studies have investigated the responses of European wild rabbit (Oryctolagus cuniculus) to predators.

Most of these experiments have used individuals under laboratory conditions, and few attempts have been made to validate the results under field conditions [START_REF] Apfelbach | The effect of predator odours in mammalian prey species: a review of field and laboratory studies[END_REF][START_REF] Monclús | Fox odour increases vigilance in european rabbits: a study under semi-natural conditions[END_REF]. For example, in a laboratory experiment, [START_REF] Monclús | Behavioural and physiology responses of naïve European rabbits to predator odour[END_REF] demonstrated that wild rabbits recognise predator odours, independently of prior experience; however, the response to fox odour under seminatural conditions was not as pronounced as under laboratory conditions [START_REF] Monclús | Fox odour increases vigilance in european rabbits: a study under semi-natural conditions[END_REF]. In another experiment under natural conditions, the grazing spatial behaviour of rabbits was not affected by manipulation of predation risk, but it influenced rabbit temporal activity pattern [START_REF] Bakker | Experimental manipulation of predation risk and food quality: effect on grazing behaviour in a central-place foraging herbivore[END_REF]; however, there is little information about the behavioural responses of rabbits at the population level in the wild.

Thus, we conducted a simple experiment to investigate the effect of previous experience on the response of a rabbit population to native predator cues in the wild. We expected that both rabbit populations, one experienced and another naïve to terrestrial predators, will respond to the scent of a predator (red fox, Vulpes vulpes) [START_REF] Monclús | Fox odour increases vigilance in european rabbits: a study under semi-natural conditions[END_REF][START_REF] Vitale | Changes in the anti-predator responses of wild rabbits, Oryctolagus cuniculus, (L), with age and experience[END_REF]).

Materials and methods

Study area and the origin of the animals

The experiment was conducted in two plots (4 ha each, see [START_REF] Rouco | The effect of exclusion of terrestrial predators on short-term survival of translocated European wild rabbits[END_REF]) separated by 1 km in Sierra Norte Natural Park of Seville (SW Spain). Eighteen artificial warrens were regularly distributed on each plot and separated 35-40 m (see Rouco et al. 2010b for further information). Water and food suppliers located close to each warren provided rabbits with water and commercial food pellets ad libitum. One of the plots was surrounded by a fence 3 m high that was topped with an electric shepherd to completely exclude terrestrial predators. Rabbits in this plot were mostly descendants of wild individuals that were translocated during autumn 2002 (Rouco et al. 2010a); thus they were therefore largely naïve to direct contact with any terrestrial predator. The second plot was unfenced, so rabbits were in contact with terrestrial predators. Red fox is the main predator in this area (average density 0.79 foxes/km 2 , authors unpublished).

Sampling design

As a source of odour, we used fresh red fox faeces collected in the study area. To make the odour uniform throughout the experiment, 500 g of faeces were stirred with 2 L of water, and the mixture was left standing for 72 h. The resulting solution (henceforth referred to as fox faeces odour) was filtered and frozen at -20°C until used.

In each plot, three treatment and three control warrens were randomly selected, and two odour stations were added to each warren; each station consisted of a chalk tied at approximately 20 cm above ground to a sheaf nailed in the ground. In the treatment warrens, the chalks were impregnated with 4 ml of fox faeces odour every 3 days during the treatment period (March to August 2004). Odour stations in the control warrens were impregnated with water following the same regime as for the treatment warrens.

To evaluate the presence of predators, and thus the predation risk perceived by the animals in the unfenced plot, counts of scats were performed weekly on an 800-m walk transect within the plot.

The monitoring of rabbit abundances and trends during the experiment was based on pellet counts [START_REF] Rouco | The effect of exclusion of terrestrial predators on short-term survival of translocated European wild rabbits[END_REF]. Pellets were counted and cleared from ten fixed circular plots (18 cm in diameter) placed randomly at 5 m far for each warren. Pellet counts were performed four times during the study period; first count in February 2004 (before treatment); second and third counts during May and August, respectively (during treatment) and last count in November (after treatment). To standardise all pellet counts, a defecation rate per day (DRD) was calculated for each count. The DRD was obtained by dividing the number of pellets at each counting station by the number of days since the last count at the same station [START_REF] Cabezas | An experimental study of translocation success and habitat improvement in wild rabbits[END_REF].

Data analysis

Pellet abundance data were analyzed using generalized linear mixed models, as implemented in SAS (SAS Institute, Inc. 2004; Genmod, Glimmix and mixed procedures) with 'warren' and 'pellet counting station' as random categorical variables. The dependent variable DRD was modelled with Poisson errors and log link. Three factors were considered in the models of pellet abundance: 'treatment' (control and treatment warrens), 'plot' (fenced and unfenced) and 'counts' (each pellet count in February, May, August and November). Denominator degrees of freedom were estimated using Satterthwaite's formula [START_REF] Littell | SAS system for mixed models[END_REF]. Nonparametric tests were used to check for differences in pellet abundance between the control and treatment warrens.

Results

No differences were found in rabbit pellet abundance between the control and treatment warrens before the treatment in either the unfenced (Mann-Whitney U test, before: Z=0.28, p=0.779) or the fenced plot (Mann-Whitney U test, before: Z=0.63, p=0.294). Rabbit population trends in both plots (unfenced and fenced) were similar (Fig. 1) and no significant differences were found in the DRD between the two plots during the study period (Table 1; plot × count). We found a significant effect of treatment (Table 1) with higher DRD in the control than in the treatment warrens in both plots (Fig. 1). The DRD value in control warrens at the end of the experiment was 4.6 times higher in the fenced than the unfenced plot (Mann-Whitney U test, Z=-3.386, p<0.001); however, after cessation of treatment, the DRD was not different between the control and treatment warrens in the unfenced (Mann-Whitney U test: Z=0.15, p=0.144) and the fenced plots (Z=-0.59, p=0.554). Predator activity in the unfenced plot increased more than fourfold during the study, as indicated by the predator scats collected along the walk transects (three in February, four in May, 18 in August and 23 in November).

Discussion

Consistent with previous studies [START_REF] Monclús | Behavioural and physiology responses of naïve European rabbits to predator odour[END_REF][START_REF] Vitale | Changes in the anti-predator responses of wild rabbits, Oryctolagus cuniculus, (L), with age and experience[END_REF]), our results indicate that rabbits respond to fox odour, even those rabbits naïve to predator cues. The main anti-predator behaviour observed was spatial avoidance, as evidenced by the lower DRD in the treatment warrens. Some animals may have left the treatment warrens due to a greater perceived predation risk. In fact, the effect of intimidation by predators seems to be more important than the consumption of prey during predator-prey interactions [START_REF] Preisser | Scared to death? The effects of intimidation and consumption in predator-prey interactions[END_REF]. The reduced response in the plot with experienced animals (unfenced plot) could be explained by the fact that the control warrens were not an ideal control because predator scats were naturally occurring nearby. This is not unexpected because fox scent marks were more common in areas of higher rabbit density [START_REF] Monclús | Red foxes (Vulpes vulpes) use rabbit (Oryctolagus cuniculus) scent marks as territorial marking sites[END_REF]. Thus, the natural presence of odour in both treatment and control warrens in the unfenced plot could reduce the effect of the treatment. For instance, [START_REF] Monclús | Behavioural and physiology responses of naïve European rabbits to predator odour[END_REF] suggested that under natural conditions, such as it occurs in our unfenced plot, rabbits can change their use of space in the presence of predator cues. Several studies have confirmed a response of mammalian prey to novel olfactory cues per se, but the response is generally stronger with predator scent marks than with non-predator odours [START_REF] Bakker | Experimental manipulation of predation risk and food quality: effect on grazing behaviour in a central-place foraging herbivore[END_REF][START_REF] Bramley | Responses of wild Norway rats (Rattus norvegicus) to predator odours[END_REF].

In the plot with the predator exclusion fence, the effect of the treatment was apparently greater at the beginning of the experiment (Fig. 1). After the initial stage, the differences in DRD between the control and treatment warrens decreased. Learnt behaviour could be modulating this response [START_REF] Griffin | Training captive-bred or translocated animals to avoid predators[END_REF], but this was less likely in the unfenced plot owing to the continuous presence of terrestrial predators.

Finally, in both the fenced and unfenced plots, we observed that the effect of the predator odour between the control and treatment warrens apparently disappeared following cessation of the treatment. [START_REF] Bakker | Experimental manipulation of predation risk and food quality: effect on grazing behaviour in a central-place foraging herbivore[END_REF] also manipulated perceived predation risks of a population of rabbits using predator odour and showed temporal avoidance of predation. It may be that after the treatment ceased and predator cues could no longer be detected, rabbits began re-occupying the warrens, suggesting the

*

  Shadow area represents the treatment period length

Fig. 1

 1 Fig. 1 Comparison of DRD (rabbit defecation rate per day) during the four pellet counts (means ± standard error) on control and treatment warrens in unfenced and fenced plot

Table 1 F

 1 values in generalized linear mixed models of the effect of treatment (control warrens vs. treatment warrens), plot (unfenced vs. fenced) and pellet counts (counts)

	Predictors	DF	F	P
	Treatment	1,358	7.05	0.008
	Plot	1,358	10.80	0.001
	Counts	1,358	4.85	0.028
	plot × count	1,357	0.43	0.514
	plot × treatment	1,357	1.29	0.257
	plot × count × treatment	1,355	0.15	0.696

Eur J Wildl Res (2011) 57:395-398

Acknowledgements C. Rouco was supported by a postdoctoral grant from the regional government of Castilla-La Mancha and the European Social Fund. Funding was provided by the projects by Confederación Hidrográfica del Guadalquivir, by the Projects CGL2009-10741, CGL2009-11665, FAU2006-0014-C-02-02, POII09-0099-2557 and PEII09-0097-436. Special thanks go to G. Calabuig, J. Castillo, A. Finque, C. Iriarte, A. Linares, S. Luna, L. E. Mínguez, O. Rodriguez and M. Reglero for their support during field work. J. Blanco-Aguilar and D. Canal provided statistical assistance, and finally thanks to M. Delibes-Mateos and one anonymous referee for their helpful comments on previous drafts of the manuscript.


Amalia-Laura Seff

Sarolta Pilbák

Ioan Silaghi-Dumitrescu

László Poppe
email: poppe@mail.bme.hu

Computational investigation of the histidine ammonia-lyase reaction: a modified loop conformation and the role of the zinc(II) ion

Keywords:

The possible reaction intermediates of the histidine ammonia-lyase (HAL) reaction were investigated within a tightly closed active site of HAL from Pseudomonas putida (PpHAL).

The closed structure of PpHAL derived from the crystal structure of PpHAL inhibited with Lcysteine, in which the 39-80 loop including the catalytically essential Tyr53 was replaced. This modified loop with a closed conformation was modeled using the structure of phenylalanine ammonia-lyase from Anabaena variabilis (AvPAL) with a tightly closed active site as a template. In this active site of HAL, three hypothetical structures of the covalently bound intermediate were investigated by conformational analysis. The distances between the acidic pro-S β-hydrogen of the ligand and the appropriate oxygen atoms of Tyr53, Ty280 and Glu414which may act as enzymic basesin the conformations of the three hypothetical intermediate structures were analyzed together with the substrate and product arrangements. The calculations indicated that the plausible pathway of the HAL reaction involved the N-MIO intermediate structure in which the L-histidine substrate was covalently bound to the apoenzyme via the amino group. Density functional theory (DFT) calculationson a truncated model of the N-MIO intermediate containing a Zn 2+ ion coordinated to the imidazole ring of the ligand and to His83, Met382 and a water moleculeindicated the role of a Zn-complex formation in the reactivity and substrate specificity of HAL.

Introduction

The degradation of histidine to glutamate follows a different pathway than that of other amino acids catalyzed by transaminases [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF] In the first stage, histidine ammonia-lyase (HAL, EC 4.3.1.3), eliminates the α-amino group from L-histidine (L-His) resulting the α,β-unsaturated (E)-urocanate (Fig. 1). The (E)-urocanic acid is a component of human sweat and has been suggested to act as a sun blocker in human skin [START_REF] Morrison | A wavelength effect on urocanic acid E/Z photoisomerization[END_REF]. The absence of HAL in humans is known as a disease called histidinemia [START_REF] Taylor | Histidase and histidinemia. Clinical and molecular considerations[END_REF].

<Figure 1>

HAL is a subset of the ammonia-lyase family together with the phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) and tyrosine ammonia-lyase (TAL, EC 4.3.1.23) catalyzing the deamination of the corresponding L-amino acid to (E)-cinnamic acid and (E)-coumaric acid [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF]. Cloning and sequencing of Pseudomonas putida genes that code HAL (hutH) and urocanase (hutU) have been described [START_REF] Consevage | Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida[END_REF][START_REF] Fessenmeier | Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida[END_REF]. HAL is also present in mammals (rat and human) [START_REF] Taylor | Localization of histidase to human chromosome region 12q22→q24.1 and mouse chromosome region 10C2→D1[END_REF][START_REF] Taylor | Identification of the mutation in murine histidinemia (his) and genetic mapping of the murine histidase Locus (Hal) on chromosome 10[END_REF][START_REF] Taylor | Site-directed mutagenesis of conserved serines in rat histidase[END_REF]. An extensive analysis of the taxonomic distribution and phylogeny of PAL/TAL/HAL revealed that eukaryotic HAL, fungi PAL and land plants PAL have distinct origins [START_REF] Emiliani | A horizontal gene transfer at the origin of phenylpropanoid metabolism: a key adaptation of plants to land[END_REF]. Taxonomic distribution of HAL and PAL orthologues indicated that the ancestor of eukaryotes harbored a HAL while a PAL was introduced by horizontal gene transfer in the ancestor of fungi and the ancestor of land plants [START_REF] Emiliani | A horizontal gene transfer at the origin of phenylpropanoid metabolism: a key adaptation of plants to land[END_REF]. Histidine ammonia-lyase from Pseudomonas putida [START_REF] Hernandez | Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli[END_REF] was found to have [START_REF] Arnold | The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[END_REF][START_REF]SWISS-MODEL[END_REF][START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF][START_REF] Langer | The role of dehydroalanine in catalysis by histidine ammonia-lyase[END_REF] and 31% amino acid sequence identity to Rattus norvegicus HAL [START_REF] Taylor | Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases[END_REF], to Bacillus subtilis HAL [START_REF] Oda | Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon[END_REF], to Anabaena variabilis (bacterial) PAL [START_REF] Wang | Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia-lyase[END_REF], to Rhodosporidium toruloides (yeast) PAL [START_REF] Calabrese | Crystal structure of phenylalanine ammonia-lyase: multiple helix dipoles, implicated in catalysis[END_REF] and to Petroselinum crispum (parsley) PAL [START_REF] Ritter | Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[END_REF], respectively.

The non-oxidative deamination of the substrates, catalyzed by HAL, PAL and TAL requires the presence of 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) electrophilic prosthetic group [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Poppe | Methylidene-imidazolone: a novel electrophile for substrate activation[END_REF][START_REF] Rétey | Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group[END_REF] in the enzyme. As a crystal structure study of three mutants (D145A, F329G and F329A) of HAL from Pseudomonas putida (PpHAL) indicated [START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF], MIO is formed autocatalytically by cyclization and dehydration of an Ala-Ser-Gly tripeptide (Fig. 2) [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Röther | Spectroscopic evidence for a 4-methylidene imidazol-5-one in histidine and phenylalanine ammonia-lyases[END_REF]. The MIO prosthetic group was later identified in the crystal structures of yeast [START_REF] Calabrese | Crystal structure of phenylalanine ammonia-lyase: multiple helix dipoles, implicated in catalysis[END_REF]21], plant [START_REF] Ritter | Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[END_REF] and bacterial PAL [START_REF] Moffitt | Discovery of two cyanobacterial phenylalanine ammonia lyases: kinetic and structural characterization[END_REF] and bacterial TAL [START_REF] Louie | Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonialyases[END_REF]. MIO was identified in Lphenylalanine and L-tyrosine-2,3-aminomutases (PAM [START_REF] Walker | Cloning, heterologous expression, and characterization of a phenylalanie aminomutase involved in taxol biosynthesis[END_REF][START_REF] Steele | Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in taxol side-chain biosynthesis[END_REF][START_REF] Mutatu | Unusual mechanism for an aminomutase rearrangement: Retention of configuration at the migration termini[END_REF] and TAM [START_REF] Christenson | A novel 4-methylideneimidazole-5one-containing tyrosien aminomutase in enediyne antitumor antibiotic C-1027 biosynthesis[END_REF][START_REF] Christianson | The structure of l-tyrosine 2,3-aminomutase from the C-1027 enediyne antitumor antibiotic biosynthetic pathway[END_REF], respectively) as well.

<Figure 2>

Histidine, phenylalanine and tyrosine ammonia-lyases should remove the non-acidic pro-S βproton from their substrates, without extracting the more acidic protons from the ammonium moiety of the corresponding L-amino acid [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF]. Biochemical data were interpreted by means of two significantly different mechanisms (Fig. 1) [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF]. The first proposals for the mechanism of the ammonia elimination catalyzed by HAL [START_REF] Smith | Inactivation of histidine deaminase by carbonyl reagents[END_REF] and PAL [START_REF] Hanson | L-phenylalanine ammonia-lyase. IV. Evidence that the prosthetic group contains a dehydroalanyl residue and mechanism of action1[END_REF] suggested that an interaction between the amino group of the substrate and the electrophilic prosthetic group of the enzyme (i.e. an N-MIO intermediate) facilitates the reaction owing to the formation of a better leaving group [START_REF] Hanson | L-phenylalanine ammonia-lyase. IV. Evidence that the prosthetic group contains a dehydroalanyl residue and mechanism of action1[END_REF][START_REF] Hermes | Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine ammonia-lyase[END_REF]. Due to the difficulty entailed by this possible mechanism to abstract the nonacidic pro-S β-proton by the enzymic base in the course of ammonia elimination, an alternative mechanism involving a Friedel-Crafts (FC) type attack at the aromatic ring of the substrates by the electrophilic prosthetic group has been suggested [START_REF] Langer | The role of dehydroalanine in catalysis by histidine ammonia-lyase[END_REF]. In spite of the significant differences in the properties of these enzymes, the presence of their similar electrophilic prosthetic group seemed to indicate that HAL, PAL and TAL catalyze their reactions by analogous mechanisms [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF].

After elucidation of the structure of HAL by X-ray crystallography and discovery of MIO (Fig. 2) [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF], the importance of several amino acid residues at the active center (Tyr53, His83, Asn195, Gln277, Tyr280, Arg283, Phe329 and Glu414) for catalysis and substrate binding was evaluated by site-directed mutagenesis [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF]. Mutagenesis was also performed for active site amino acid residues of PAL [START_REF] Röther | An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum[END_REF] that were identical or similar to the active site residues of HAL. The almost total loss of activity observed with the mutant Y53F of HAL (2650 times less active than the wild-type HAL) and with the mutant Y110F of PAL (75 000 times less active than the wild-type PAL) demonstrated that Tyr53 in HAL and Tyr110 in PAL were essential for the catalysis [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF][START_REF] Röther | An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum[END_REF].

A unique feature of the reaction catalyzed by HAL is the significant enhancement of the enzyme activity in the presence of divalent metal ions, such as Zn 2+ , Cd 2+ or Mn 2+ [START_REF] Klee | Metal activation of histidine ammonia-lyase. Metal ion-sulfhydryl group relationship[END_REF]. The optimal activity of HAL was shown to be dependent on the addition of metal ions [START_REF] Klee | Metal activation of histidine ammonia-lyase. Metal ion-sulfhydryl group relationship[END_REF][START_REF] Givot | Studies on the mechanism of action and the structure of the electrophilic center of histidine ammonia lyase[END_REF].

Based on these experimental data, it was already proposed that the interaction of the substrate with the His83 imidazole group could be mediated by coordination with Zn 2+ [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF].

Although mutagenesis data [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF] and six crystal structures [16, 19 37] are available for HAL, the nature of the covalent reaction intermediate (Fig. 1) and the role of Zn 2+ ion in the 5 catalysis have not been explored or investigated by systematic computations. It has been demonstrated alreadyin the case of the partially modified parsley phenylalanine ammonialyase (PcPAL)that homology modeling, systematic conformational search and ligand docking can be applied for mechanistic studies for an ammonia-lyase [START_REF] Röther | An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum[END_REF][START_REF] Seff | Ligand docking and systematic conformational analysis in loop modified parsley phenylalanine ammonia-lyase structure[END_REF][START_REF] Bartsch | A single residue influences the reaction mechanism of ammonia lyases and mutases[END_REF]. Based on the above data and methods, our goal in this study is to explore the structure of the covalent intermediate and the role of Zn 2+ in the HAL reaction by using partial homology modeling, systematic conformational search and DFT calculations.

Theory and methods

Experimental ammonia-lyase structures used for the computations

Six crystal structures of Pseudomonas putida histidine ammonia-lyase HAL (Brookhaven Protein Data Bank (PDB) codes: 1B8F, unliganded PpHAL Cys273Ala-mut [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF]; 1EB4, unliganded PpHAL Phe329Ala-mut [START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF]; 1GK2, unliganded PpHAL Phe329Gly-mut [START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF]; 1GK3, unliganded PpHAL Asp145Ala-mut [START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF]; 1GKJ, unliganded PpHAL Tyr280Phe-mut [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]; 1GKM, PpHAL inhibited with L-cysteine [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]) were compared. The six HAL structures showed only small differences in the positions of the amino acid side chains within the active center, with the exception of the arrangement of Met382 in the PpHAL inhibited with L-cysteine (1GKM).

Modeling the Tyr53-containing 39-80 loop region of PpHAL structure

A HAL structure with a closed active site (1GKM mod ) was constructed by modifying the PpHAL structure which included L-cysteine inhibitor (PDB code: 1GKM) by the replacement of the 39-80 loop, containing the catalytically essential Tyr53, in every subunit of the homotetrameric enzyme. The crystal structure of Anabaena variabilis phenylalanine ammonia-lyase PAL (PDB code: 3CZO) with a tightly closed active site [START_REF] Wang | Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia-lyase[END_REF] has been used as a template for modeling the closed conformation of the 39-80 loop of PpHAL. For homology modeling, the SwissPDB Viewer [START_REF]Swiss-PDBViewer version 4.0[END_REF][START_REF] Guex | SWISS-MODEL and the Swiss-Pdb Viewer: An environment for comparative protein modeling[END_REF][START_REF] Schwede | SWISS-MODEL: an automated protein homology-modeling server[END_REF] program suite and the Swiss-Model automated homology modeling service [START_REF]SWISS-MODEL[END_REF][START_REF] Arnold | The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[END_REF][START_REF] Kopp | The SWISS-MODEL repository of annotated threedimensional protein structure homology models[END_REF][START_REF] Peitsch | Protein modelling by e-mail[END_REF] were used.

Construction of the covalently bound MIO-substrate intermediate models in the 1GKM mod structure

The L-cysteine inhibitor (Cys1510) within the modified structure of PpHAL (1GKM mod ) was replaced by L-histidine. A portion of the 1GKM mod structure (overlapping spheres of 15 Å radius around the C 1 of the ligand and 12 Å radius around the O atom of the W544 water, consisting of 149 amino acid residues) was used as the active site model of PpHAL for CS calculations. Three initial ligand structures [an N-MIO model and two diastereomeric (S,S)-FC and (R,S)-FC models] were built for the systematic conformational analysis search (CS) from this L-histidine by turning its carboxyl group towards Arg283 and its imidazole ring close to the HAL-specific His83 residue and constructing the appropriate bonds between the methylene carbon of MIO and the nitrogen atom of amino group or the C 4 atom of imidazole of the ligand. Then, hydrogens were added to the model portion of the 1GKM mod structures and the C-and N-termini at cuts were transformed into neutral aldehyde and amino moieties by HyperChem [START_REF]HyperChem version 7.0 (Hypercube, Inc[END_REF] standard procedure. The resulted structures were verified and corrected using Molfunction [START_REF] Molfunction | Institute of Molecular Function[END_REF]. varied during the CSs. The CSs were performed by the HyperChem implemented CS module [START_REF]HyperChem version 7.0 (Hypercube, Inc[END_REF] using the default settings (MM+ forcefield; gradient: 0.1 kcal mol -1 ; Polak-Ribiere method; limits: 300 iterations, 150 optimizations, 15 conformations; test options: "skip if atoms are closer than 0.3 Å").

Conformational analysis for the covalently bound MIO

Geometry optimization of the covalent intermediates, L-histidine and (E)-urocanate within the active site of HAL

-Histidine and (E)-urocanate structures were built, by proper rearrangement of bonds (Fig. 3), from those conformers of the covalent intermediates which had straight main-chain conformation [N-MIO series (N : c1-c3, c5, c8, c9, c11; N : c1-c4, c6, c9, c10); (R,S)-FC series (N : c2, c4, c5, c7-c9; N : c2) and (S,S)-FC series (N : c1-c2; N : c2)]. In all substrate, intermediate and product structures, the ligand together with MIO (substrate, intermediate) or NH 2 MIO (product) and the His83, Tyr280, Tyr53, Asn195, Gln277, Glu414, Arg283 and Phe329 (a total of 116 atoms) were optimized in a 15 Å spherical part (2221 atoms) of the 1GKM mod structure, while keeping the residual part rigid. The optimizations were performed by the HyperChem [START_REF]HyperChem version 7.0 (Hypercube, Inc[END_REF] implemented MM+ forcefield with the default settings (gradient: 0.1 kcal mol -1 ; Polak-Ribiere method).

<Figure 3>

Docking L-histidine and (E)-urocanate into the 1GKM mod active site

The -cysteine inhibitor and water molecules were removed from the 1GKM mod structure and a docking environment containing 29 amino acid residues [including His83, Arg283, Met382, Tyr53, Tyr280, Asn195 and Glu414 and MIO] was selected. For docking zwitterionichistidine, Tyr53 and Tyr280 were kept deprotonated and four torsion angles (along the axes of C 5 -C , C -C , C -N and C -C COO -) were varied, whereas for docking (E)-urocanate, protonated forms of Tyr53 and Tyr280 were used. Gasteiger charges were added to the atoms of the binding interfaces used for docking by AutoDock [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF][START_REF] Huey | A semiempirical free energy force field with charge-based desolvation[END_REF]. During the docking with AutoDock [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF][START_REF] Huey | A semiempirical free energy force field with charge-based desolvation[END_REF], the zwitterionic L-histidine and (E)-urocanate were flexible in a rigid enzymic environment. The Grid Parameter File was established with the default settings (Total Grid Pts per map: 77326; number of points 40 for x-and y-dimensions, 45 for zdimension; spacing: 0.375 Å; center on ligand) and docking processes were also run with the default settings (Lamarckian Genetic Algorithm: 10 runs; population size: 150; maximum number of energy evaluations: short setting).

DFT calculations on ligands involved in HAL reactions

DFT calculations were carried out on -histidine and -4-nitrohistidine models (both with protonated amino groups) for conformations corresponding to the HAL bound state, on a truncated model of the N-MIO-intermediate (by replacing the MIO ring of the calculated structure with a hydrogen atom at the exocyclic methylene carbon of MIO) and on a partial active site model including the truncated model of the N-MIO-intermediate in coordination with a Zn 2+ ion, which is also coordinated to representative parts of His83 and Met382 and to a water. The DFT optimizations were performed using the 09 (rev. A.1) [START_REF] Frisch | Gaussian 09, Revision A.1. Gaussian Inc[END_REF] and GaussView [52] as front-end. Two positively charged -histidine structures (with -COOH and -NH 3 + ) were constructed from the N-MIO models (from conformations c5 and c4 of the N τH and N πH series of CSs, respectively). Two positively charged -nitrohistidine models were built from these two -histidine structures by replacing the hydrogen at C 4 of the imidazole with a nitro group.

In the Zn 2+ complex models, the Zn 2+ ion had four or five ligands: the imidazole of the

N

Results and discussion

Unfortunately, the available X-ray structures of HAL do not contain substrate, product or Zn 2+

ion [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]. This may be due to the partially open active site of the available HAL structures (Panel a in Fig. 4). Therefore, the goals of this study were to generate a proper HAL enzymic environment to investigate the alternative enzyme-bound reaction intermediates and the role of Zn 2+ ion by means of computations.

Construction of a closed HAL active site environment for calculations

For modeling the closed active site containing state of HAL, the L-cysteine-inhibited PpHAL [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF] was selected as a starting structure (PDB code: 1GKM). This is the only crystal structure of PpHAL in which an inhibitor is present. Importantly, this is also the structure where the side chain of Met382 has a different conformation from the side chain arrangement of the other five unliganded PpHAL structures [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF].

Although the existing six PpHAL structures contain the Tyr53 inside the active site [START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF], a structural comparison of HALs to the ammonia-lyases with tightly closed active sites indicated the Tyr-loops in all HAL structures are in a partially open conformation (Fig. 4a).

The importance of the loop containing the catalytically essential tyrosine in the MIOcontaining ammonia-lyases was best demonstrated for PAL. The Tyr-loops were missing [START_REF] Calabrese | Crystal structure of phenylalanine ammonia-lyase: multiple helix dipoles, implicated in catalysis[END_REF]21,[START_REF] Moffitt | Discovery of two cyanobacterial phenylalanine ammonia lyases: kinetic and structural characterization[END_REF] or were in catalytically inactive conformation [START_REF] Ritter | Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[END_REF][START_REF] Pilbák | The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases[END_REF] in several crystal structures of PAL. Only recently, the structure of PAL from Anabaena variabilis (AvPAL) containing a tightly closed active site confirmed the active conformation of the Tyr-loop [START_REF] Wang | Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia-lyase[END_REF]. Similarly, the crystal structure of Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) revealed a tight active center in which the loop containing the essential Tyr60 was present in active conformation [START_REF] Louie | Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonialyases[END_REF].

Comparison of the PpHAL (1GKM, in blue) structure to RsTAL (2O7B, in green) [START_REF] Louie | Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonialyases[END_REF] and AvPAL (3CZO, in orange and CPK color) [START_REF] Wang | Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia-lyase[END_REF] with compact active centers revealed that the catalytically essential Tyr53-containing loop of PpHAL adopts a partially open conformation 10 (Fig. 4a). Because the most compact structure has been found for AvPAL (3CZO, 2.2 Å resolution) containing the non-solvent accessible essential Tyr78 and the electrophilic MIO deeply buried in the active center [START_REF] Wang | Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia-lyase[END_REF], this ammonia-lyase structure has been selected as a template for modeling the compact conformation of the Tyr-loop of PpHAL.

Homology modeling was successful for modeling the essential Tyr-containing loop region of PAL (PcPAL, PDB code: 1W27 [START_REF] Ritter | Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[END_REF]) and thus in creating a proper active site environment for calculations inside PAL [START_REF] Seff | Ligand docking and systematic conformational analysis in loop modified parsley phenylalanine ammonia-lyase structure[END_REF][START_REF] Pilbák | The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases[END_REF]. Therefore, a similar strategy was used to construct the

Comparison of the conformation of the covalent reaction intermediates of the HAL reaction with the arrangements of the substrate and product

Some early reports have indicated that treatment of HAL at high pH in the presence of Lcysteine and oxygen leads to an irreversible inactivation of the enzyme [START_REF] Klee | Stereospecific irreversible inhibition of histidine ammonia-lyase by Lcysteine[END_REF][START_REF] Hernandez | Identification of Ser 143 as the site of modification in the active site of histidine ammonia-lyase[END_REF]. On denaturation, the L-cysteine inhibited HAL, followed by pronase digestion resulted in two main chromophoric products [START_REF] Merkel | Further insight into the mechanism of the irreversible inhibition of histidine ammonia-lyase by L-cysteine and dioxygen[END_REF]. In one product, the exocyclic methylene of the MIO was substituted by the amino groups of L-cysteine. When L-cysteine inhibited HAL was first digested with trypsin, two chromophoric 24-residue peptides were isolated and identified as N MIO fragments [START_REF] Galpin | The inactivation of histidine ammonia-lyase by L-cysteine and oxygen: modification of the electrophilic center[END_REF]. This was later supported by the L-cysteine inhibited structure of PpHAL (PDB code: 1GKM) [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]. The inhibited HAL contains the inhibitor with its amino moiety close to the exocyclic methylene of the electrophilic MIO prosthetic group. This fact can be considered as a further proof for the presence of an amino-enzyme intermediate in the HAL reaction demonstrated by Peterkofsky [START_REF] Peterkofsky | The mechanism of action of histidase: Amino-enzyme formation and partial reactions[END_REF].

Structures of the HAL with L-cysteine [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF], TAL with 2-aminoindan-2-phosphonate inhibitor [START_REF] Louie | Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonialyases[END_REF] and TAM co-crystallized with , -difluoro-β-tyrosine [START_REF] Christianson | The mechanism of MIO-based aminomutases in β-amino acid biosynthesis[END_REF] or p-fluorocinnamate epoxide [START_REF] Montavon | Design and characterization of mechanism-based inhibitors for the tyrosine aminomutase SgTAM[END_REF] provided strong evidence for reactions via N MIO intermediates (in which the substrate is connected to MIO through its amino group) for the ammonia-lyase and aminomutase

reactions. An alternative covalently bound intermediate was proposed by Rétey and coworkers [START_REF] Langer | The role of dehydroalanine in catalysis by histidine ammonia-lyase[END_REF]. In this case, a σ-complex would be formed between the aromatic part of the substrate and the MIO prosthetic group by Friedel-Crafts-like mechanism (FC).

Analysis of the active site residues surrounding the ligand in the L-cysteine-inhibited HAL [START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF] indicated that the pro-S -proton from the -histidine substrate can be abstracted by one of the three residues (Tyr53, Tyr280, Glu414) which might be considered as enzymatic bases (Fig. 5). Mutagenesis experiments also demonstrated that Tyr53, Glu414 [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF], and Tyr 280 [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF] are important residues for the catalysis. The remarkably reduced catalytic activity of the analogous tyrosine (Tyr 60 and Tyr300) mutants of TAL [START_REF] Schroeder | Contribution of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase[END_REF] implies the importance of Tyr53 and Tyr280 in the HAL reaction.

When PAL was investigated with the phenylalanine analogues D-and L-2-aminooxy-3phenyl-propionic acid, it was deduced that ammonia elimination approximated the leastmotion course [START_REF] Hanson | Phenylalanine ammonia-lyase: mirror image packing of D-and Lphenylalanine and D-and L-transition state analogs into the active site[END_REF]. In several RsTAL structures, products of the elimination reaction were found in the active site in a zig-zag orientation [START_REF] Louie | Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonialyases[END_REF]. The least-motion course principle and the similar straight chain zig-zag shape of the (E)-urocanate product of the HAL reaction determine a straight chain zig-zag arrangement of the covalently bound intermediate and the L-histidine substrate as well. Irrespectively of the nature of the covalently bound reaction intermediate, four reaction paths can fulfill these requirements (Fig. 5).

<Figure 5>

Along the A-1 reaction path for transformation of the substrate to product, both N MIO and FC intermediates are possible but only the Tyr53 amino acid could abstract the pro-S β-proton from the substrate. Only an FC-like mechanism is possible via the A-2 path involving Tyr280 as the base for pro-S β-proton abstraction. Along the B-1 path, deamination of -histidine may take place by the FC mechanism involving Glu414 as an enzymic base. Along the B-2 path, both types of the mechanism (N MIO and FC) can be taken into account involving Tyr53 as the base for abstraction of the pro-S β-proton.

In addition to the substrate and product states (Fig. 5), the covalently bound reaction kcal mol -1 range (Table 1). From the retained conformations of the covalent intermediates of the six CSs, -histidine and (E)-urocanate containing active site models were constructed. The substrate and product and the surrounding eight catalytically relevant amino acid residues were optimized within the closed HAL (1GKM mod ) active site. In this way, comparative analysis of the full S → I → P(+NH 2 ) reaction pathways become feasible (Table 1).

Although the energies obtained at the MM level of theory are usually not accurate enough for reliable enzyme mechanistic studies, several observations are worth mentioning. According to our calculations (Table 1), energetic results also favor the reaction via N MIO (N H ) type 13 intermediates (especially via the c5 and c8 conformations). In the N MIO (N H ) case, the substrate binding states are of substantially higher energy than the corresponding reactive N MIO type intermediates and the product binding states are of the lowest energies. As in all cases of the N MIO (N H ) type route the energy of the substrate biding state is substantially higher than the energy of the corresponding product biding state, the calculated energy profile of the reaction is in full agreement with experimental results since MIO-containing ammonialyases catalyze, under normal conditions, ammonia elimination from the L-amino acids in a practically irreversible manner [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF][START_REF] Rétey | Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group[END_REF]. Although the other types of intermediates can be disclosed as reactive species by non-energetic arguments discussed later, it is worth to note thet all the other intermediate structures had higher energies (10.8-33.1 kcal mol -1 ) than the lowest energy N MIO (N H ) intermediate conformer.

Next, the H-O distances in the optimized structures of the MIO-bound intermediate models between the ligand's pro-S -hydrogen and oxygen atoms of the possible enzymic bases (Tyr53, Tyr280 and Glu414) and between ligand's carboxylate oxygen group and Arg283

were analyzed (Table 1). The most decisive result of this analysis was the observation that there was no enzymic base in any of the FC-intermediate conformations which was close enough to the pro-S -hydrogen to abstract it. Therefore, the A-1 pathway involving an N MIO covalent intermediate is the most plausible for the HAL reaction but a B-2 type orientation would also be allowed.

<Table 1>

The optimized structures containing L-histidine and (E)-urocanate ligands in the active site of the closed HAL structure were compared to the arrangements of these ligands obtained by a docking procedure. The analysis of the AutoDock results revealed a well conserved orientation of the product (the carboxylate of the ligand is in the vicinity of the Arg283 while the imidazole moiety points towards His83) and agreed with the results obtained from the MM optimizations. Taking into account the orientation of the product within the active site, the docking results corresponded only to the A-1 pathway.

The role of Zn(II) in the HAL reaction

The first known zinc-containing enzyme, carbonic anhydrase, was discovered in 1932 and was found to contain bound zinc, associated with catalytic activity [START_REF] Meldrum | Carbonic anhydrase. Its preparation and properties[END_REF]66]. Nowadays, many enzymes are known to contain zinc coordinated to two or three histidine residues and to other residues or water molecules [START_REF] Glusker | Structural aspects of metal liganding to functional groups in proteins[END_REF][START_REF] Rulísek | Coordination geometries of selected transition metal ions (Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , and Hg 2+ ) in metalloproteins[END_REF][START_REF] Zheng | Data mining of metal ion environments present in protein structures[END_REF]. Enzymes may contain two histidines both coordinating the Zn ion by their N atoms [START_REF] Nauton | Structural insights into the design of inhibitors for the L1 metallo-β-lactamase from Stenotrophomonas maltophilia[END_REF][START_REF] Lipscomb | Recent advances in zinc enzymology[END_REF] or at their N atoms 1 or in some cases the Zn ion is coordinated by one His at N , whereas by the other His at N [START_REF] Nauton | Structural insights into the design of inhibitors for the L1 metallo-β-lactamase from Stenotrophomonas maltophilia[END_REF][START_REF] Gerhardt | Crystal structures of human ADAMTS-1 reveal a conserved catalytic domain and a disintegrin-like domain with a fold homologouse to cysteine-rich domains[END_REF][START_REF] Bebrone | Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF][START_REF] Debela | Structural basis of the zinc inhibition of human tissue Kallikrein 5[END_REF].

It was observed that Zn 2+ or a number of different divalent cations, like Cd 2+ or Mn 2+ , increase the activity of HAL [START_REF] Klee | Metal activation of histidine ammonia-lyase. Metal ion-sulfhydryl group relationship[END_REF]. On the other hand, there is no Zn 2+ containing crystal structure for HAL [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]. This apparent contradiction can be resolved by assuming that Zn 2+ , which is necessary for the catalytic activity, interacts during the HAL reaction with the HAL-specific His83 residue and with the imidazole of the substrate [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF]. Therefore, the reason why no Zn 2+ containing HAL structure is known is that no substrate or product containing HAL structure has been determined so far [START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF]. Interaction of a Zn 2+ with the HAL-specific His83 and with the substrate during the catalysis [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF] can also rationalize why HAL accepts only L-histidine [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF], L-4-fluorohistidine [START_REF] Klee | Histidine ammonia-lyase. The use of 4fluorohistidine in identification of the rate determining step[END_REF] or L-4-nitrohistidine [START_REF] Langer | The role of dehydroalanine in catalysis by histidine ammonia-lyase[END_REF][START_REF] Klee | 4-Nitro-L-histidine as a substrate for histidine ammonia-lyase: the role of beta-hydrogen acidity in the rate-limiting step[END_REF] as substrates.

<Figure 6>

To examine the contribution of the Zn 2+ in HAL, model studies were performed on the conformations found for the N-MIO intermediate in the 1GKM mod active site. Analysis of the existing Zn-containing protein crystal structures [START_REF] Nauton | Structural insights into the design of inhibitors for the L1 metallo-β-lactamase from Stenotrophomonas maltophilia[END_REF][START_REF] Lipscomb | Recent advances in zinc enzymology[END_REF][START_REF] Gerhardt | Crystal structures of human ADAMTS-1 reveal a conserved catalytic domain and a disintegrin-like domain with a fold homologouse to cysteine-rich domains[END_REF][START_REF] Bebrone | Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF][START_REF] Debela | Structural basis of the zinc inhibition of human tissue Kallikrein 5[END_REF] and the calculated N-MIO intermediate conformations led to the conclusion that Zn could be coordinated at N atoms of His83 and of the substrate's imidazole (Fig. 6a , similarly as found in Adamalysin II, a zinc endopeptidase from the snake venom of Crotalus adamanteus [START_REF] Gomis-Rüth | First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases[END_REF] (Fig. 6b).

In the Zn-containing protein structures, two kinds of Zn-complexes can be found [START_REF] Bebrone | Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF][START_REF] Debela | Structural basis of the zinc inhibition of human tissue Kallikrein 5[END_REF]. The Zn may be present in tetrahedral (Th) [START_REF] Bebrone | Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily[END_REF][START_REF] Debela | Structural basis of the zinc inhibition of human tissue Kallikrein 5[END_REF] or trigonal bypiramidal (Tbp) [START_REF] Nauton | Structural insights into the design of inhibitors for the L1 metallo-β-lactamase from Stenotrophomonas maltophilia[END_REF][START_REF] Lipscomb | Recent advances in zinc enzymology[END_REF] complexes. During the HAL reaction, the further ligands of the Zn 2+ coordinated to His83 and to the imidazole of L-histidine can be the S atom of the Met382, which is a conserved residue of the histidine ammonia-lyases, and one (in a tetrahedral Zn-coordination, Th) or two water molecules (in a trigonal bipyramidal Zn-coordination, Tbp).

The DFT calculations were performed on the two kinds of truncated Zn-complex structures (Th and Tbp) including all important parts of the active site of HAL (partial elements of MIO, His83, Met382 and one or two conserved water molecules). The DFT calculations indicated that the reaction of HAL should include a tetrahedral Zn-complex, because only tetrahedral complexes resulted in reasonable structures (Table 2). Optimizations of all possible kinds of trigonal bipyramidal Zn-complex arrangements aborted or led to tetrahedral structures by exclusion of a water. Comparison of the tetrahedral Zn-complex (Fig. 6c) with the Zn 2+ complex found in Adamalysin II [START_REF] Gomis-Rüth | First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases[END_REF] (Fig. 6b) and with the possible arrangement of the substrate-MIO covalent intermediate obtained by conformational analysis in the Zn-free active site (Fig. 6a) indicated good agreement in the spatial arrangement of the structures.

<Table 2>

The overlay of the truncated tetrahedral Zn-complex and the ligand-free HAL structure (Fig. 6c) indicated that N atom of the ligand's imidazole was involved in the Zn coordination, whereas the hydrogen atom on the N position could be at H-bond distance from Glu414 (Fig. 6a and Fig. 6c). The binding of the ligand during the HAL reaction at its imidazole ring by His83 via the Zn-complex and by Glu414 via a hydrogen bond is in full agreement with active site mutation data indicating that the Glu414Ala (k cat /k cat-mut = 20930) and the His83Leu (k cat /k cat-mut = 18000) mutations have the most dramatic effect on catalysis [START_REF] Röther | Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida[END_REF]. Although it might be considered that zinc(II) activates the enzyme by other mechanism, this mode of substrate binding can rationalize the very narrow substrate specificity (in addition tohistidine, only L-4-fluorohistidine [START_REF] Klee | Histidine ammonia-lyase. The use of 4fluorohistidine in identification of the rate determining step[END_REF] and -4-nitrohistidine [START_REF] Langer | The role of dehydroalanine in catalysis by histidine ammonia-lyase[END_REF][START_REF] Klee | 4-Nitro-L-histidine as a substrate for histidine ammonia-lyase: the role of beta-hydrogen acidity in the rate-limiting step[END_REF] are accepted as substrates [START_REF] Poppe | Friedel-Crafts type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine[END_REF]) of HAL as well.

All the previous results implied that the enzymic base in the HAL reaction which abstracts the pro-S β-hydrogen as a proton is Tyr53. This was indicated by the 1.99 Å distance between the oxygen atom of Tyr53 and the pro-S β-hydrogen of the tetrahedral Zn-complex as well (Fig. 6c).

Although Mulliken charges cannot be used as clear arguments for the acidity of hydrogen atoms, analysis of Mulliken charges of the pro-S β-hydrogens of L-histidine, L-4-nitrohistidine and truncated models for the Zn-free and Zn-coordinating N-MIO intermediates in the conformations allowed in the closed active site of HAL was performed (Table 3). L-4-Nitrohistidine was also included because it is known that the nitro group acidifies the pro-S βhydrogen of L-4-nitrohistidine [START_REF] Klee | 4-Nitro-L-histidine as a substrate for histidine ammonia-lyase: the role of beta-hydrogen acidity in the rate-limiting step[END_REF] and it is accepted as substrate even by the MIO-less mutant of HAL 32]. The Mulliken charges found for pro-S β-hydrogen at the optimized geometries of L-histidine, L-4-nitrohistidine (N τH forms) and the Zn-complex models (Table 3) indicated that the pro-S β-hydrogen was significantly more polarized in L-4-nitrohistidine 16 (Entry 3) than in the L-histidine (Entry 2) or in the Zn-free N MIO model (Entry 1). The most charged pro-S β-hydrogens, however, were found in the Zn-complex models (Entries 4-7).

<Table 3>

These results imply that the formation of a transient Zn-complex in the HAL reaction contributes not only to the specific binding of the substrate but to the enhancement of its reactivity as well. Further details of the HAL reaction and the Zn-complex will be studied on a QM/MM model of the full active site of HAL in the near future.

Conclusions

The present study revealed that the existing experimental structures of histidine ammonia-

Figure captions

Fig. 1 The histidine ammonia-lyase (HAL) reaction with the proposed alternative reaction intermediates. [The nitrogens in the imidazole rings of L-histidine and (E)urocanate are denoted according to IUPAC with pros ("near", π) and tele ("far", τ)]

  -substrate intermediates within the partial 1GKM mod structure Two separate CSs were performed for each covalently bound reaction intermediate [N-MIO, (S,S)-FC and (R,S)-FC models] bearing a proton on either the or the nitrogen of the imidazole ring (N and N series of conformers). The six CS calculations involving the ligand and MIO [28 atoms in the N-MIO model and 29 atoms in the (S,S)-FC / (R,S)-FC) models] were performed in a rigid enzymic environment without water molecules. For the N MIO model, three torsions [along the axes of C (MIO-C5) -C (MIO-CH2) , C (MIO-CH2) -N (HisL) , N (HisL) -C (HisL-C ) ], and for the (S,S)-FC and (R,S)-FC models four torsions [along the axes of C (MIO-C5) -C (MIO-CH2) , C (MIO-CH2) -C (HisL-C4) , C (HisL-C5) -C (HisL-C ) and C (HisL-C ) -C (HisL-C ) ] were

  MIO (N πH ) ligand at the N τ atom (i.e. the c4 conformer of the N MIO N πH series, with a -NH 2 (CH 3 ) + group as a model of the amino moiety bound to MIO, truncated between the MIO ring and the exocyclic carbon), a 4-methyl-1H-imidazole (coordinated at N τ , representing His83), a dimethyl sulfide (coordinated at its S-atom, representing Met382) and one (in the tetrahedral case) or two (in the trigonal bipyramidal case) water molecules. Proper constraints were used to maintain the conformation of the MIO-bound histidine ligand (HisL) as allowed within the HAL active site. For the Zn 2+ complex models, several atomic positions were frozen [in Model 1: an oxygen atom of carboxylic acid moiety of the MIO-bound histidine ligand (the one which was closer to Arg283), the carbon atom of the methyl group of 4methylimidazole (truncated His83); in Model 2: as in Model 1 + a carbon atom of the dimethyl sulfide (representing the C atom of Met382); in Model 3: as in Model 1 + methylene carbon atom of MIO; and in Model 4: as in Model 1 + a carbon atom of the dimethyl sulfide (representing the C atom of Met382) and methylene carbon atom of MIO]. Full geometry optimizations for the two types of -histidine, -4-nitrohistidine and the tetrahedral or trigonal bipyramidal Zn 2+ complexes were carried out by the DFT method using Becke's three parameter hybrid functional combined with the Lee-Yang-Parr correlation functional (B3LYP) [53, 54], with the 6-31G or 6-31G(d,p) basis sets. After optimizations, vibrational frequencies were computed at the same level of theory and single point energies were calculated with a larger basis [6-311+G(d,p) for Zn 2+ , for the two imidazole rings, for 9 the -S-CH 3 part of Met382 and for the water molecule(s); and 6-31G(d) for the other parts of the Zn 2+ complex models].

  Fig.4b, in red) to the Tyr-loop of the original L-cysteine inhibited PpHAL (1GKM, Fig.4b, in blue) indicated that the catalytically essential Tyr53 in the modified structure was closer to MIO, and therefore could better facilitate the pro-S β-proton elimination from the substrate, than in the original HAL structure. Moreover, Ramachandran-plot analysis of single subunits of the two HAL structures revealed that there were 8 amino acid residues outside the likely Phi/Psi combinations in the original HAL structure (1GKM, Fig.4c), whereas there were only 4 unlikely combinations in the Tyr53-loop of the modified HAL structure (1GKM mod , Fig.4d).

  intermediate should also fulfill the requirements of least-motion course in the HAL active site.If the HAL reaction proceeded via the N-MIO intermediate, the amino moiety of the Lhistidine substrate would be bound to MIO. If the HAL reaction proceeded via a Friedel-Crafts type intermediate, the C 4 carbon of the aromatic ring of L-histidine substrate would form a σ-complex with MIO. However, in this case the reaction may take place via two diastereomeric intermediates [(S,S)-FC and (R,S)-FC] due to a newly forming center of asymmetry at the C 4 carbon of the aromatic ring in the σ-complex.As in cases of all possible intermediates the substrate was anchored to the enzyme by a covalent bond, the systematic conformational search (CS) with the alternative reaction intermediates was a powerful tool to find their possible arrangements within the HAL active site. Because none of the six HAL[START_REF] Schwede | Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile[END_REF][START_REF] Baedeker | Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase[END_REF][START_REF] Baedeker | Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism[END_REF] crystal structures indicated significant variations at the most part of the active center, the CSs were performed in rigid enzyme environment. This approach was also supported by the analysis of the B-factors of the active site amino acid residues indicating low mobility (with exception of the residues of the mobile Tyr53-loop).Because the imidazole ring allowed two different protonation states (N or N ) for each reaction intermediates, six CSs were performed for the three principal alternatives [N-MIO-N , N-MIO-N , (R,S)-FC-N , (R,S)-FC-N , (S,S)-FC-N , (S,S)-FC-N ]. Only those conformations were kept which had a straight chain zig-zag arrangement within the lowest 10

  lyase from Pseudomonas putida (PpHAL) contain an essential Tyr53-containing loop in a partially opened conformation. The modified, closed HAL structure (constructed by modeling the 39-80 loop with the catalytically essential Tyr53 on the basis of AvPAL) resulted less deviations from the allowed side chain conformations in the Ramachandran-plot than the original experimental structure. Investigation of distances between the acidic pro-S β-hydrogen at C 2 of ligand and the appropriate oxygen atoms of the possible enzymic bases Tyr53, Ty280 and Glu414 in the calculated conformations of the three proposed structures [N-MIO, (R,S)-FC, (S,S)-FC] of the covalently bound reaction intermediate within the closed active site of HAL revealed that the reaction can only take place via the N-MIO intermediate structure which allowed Tyr53 to get close enough to the pro-S β-hydrogen. This conclusion was also supported by the docking results with (E)-urocanate. DFT calculations on the role of a Zn 2+ ion in the HAL reaction using a truncated model of the N-MIO intermediate indicated the formation of a tetrahedral complex with a Zn 2+ ion coordinated to the imidazole ring of the ligand, to His83 and Met382 residues of the enzyme and to a water molecule. The formation of such transient Zn-complex could explain the narrow substrate specificity of HAL. The DFT calculations indicated also that the formation of a Zn-complex had a contribution to the enhancement of the pro-S β-hydrogen's reactivity in the N-MIO intermediate as well.

  c n denotions refer to the covalent intermediate conformations obtained by calculations described in the "Conformational analysis for the covalently bound MIO-substrate intermediates ..." section b The methods for optimization of the substrate (S), intermediate (I) and product (P+NH 2 ) states are described in the "Geometry optimization of the covalent intermediates, L-histidine and (E)-urocanate ..." section c The types of arrangements (according to Fig. 5) are shown only for those S → I → P(+NH 2 ) reaction pathways which satisfy the least-motion principle d Distances between the ligand's carboxylate-O and the two N-H's of Arg283, and between the pro-Shydrogen of the intermediate (I) and the O -atoms of the possible enzymatic bases Tyr53/Tyr280/Glu414. Distances which are shorter than 2.5 Å are emphasized by typing in bold

Fig. 2 Fig. 3 Fig. 5 Fig. 6 a.

 2356 Fig. 2Proposed autocatalytic formation of MIO from the tripeptide Ala-Ser-Gly

Table 1

 1 MM relative energies of the models of substrate (E S ), intermediate (E I ) and product with MIO-bound amino group (E P+NH2 ) and representative H-O distances in the covalent

	Tables
	substrate-MIO intermediate structures
	Covalent intermediate a	Relative energies b (kcal/mol)	Reaction path c	H-O distances (Å) in I d
		E S	E I	E P+NH2		R283	Y53	Y280	E414
	N MIO (N H )
	c 1	14.0	7.8	-10.1	A-1	2.19/3.12	2.31	5.17	6.14
	c 2	5.6	2.7	-7.5	A-1	3.73/5.20	2.29	5.09	5.43
	c 3	14.0	8.3	-10.7	A-1	2.19/3.03	2.33	5.23	6.50
	c 5	13.9	0.2	-10.0	A-1	2.28/3.08	2.36	5.21	6.45
	c 8	5.7	0.0	-10.7	A-1	4.37/6.09	2.37	5.30	5.64
	c 9	14.7	7.8	-5.4	B-2	8.41/7.45	2.13	4.25	4.83
	c 11	21.8	15.4	-5.8	A-1	2.36/2.89	2.15	5.13	6.20
	N MIO (N H )
	c 1	20.4	11.3	14.9	A-1	4.08/5.36	2.45	5.04	5.28
	c 2	40.4	10.8	0.4	A-1	3.74/5.20	2.34	5.12	5.44
	c 3	24.1	17.9	8.5	A-1	2.18/3.12	2.28	5.16	6.14
	c 4	24.0	17.6	6.2	A-1	2.19/3.03	2.31	5.15	6.24
	c 6	24.2	19.7	5.5	A-1	2.19/2.69	2.44	5.12	6.28
	c 9	31.7	24.1	8.2	A-1	2.32/2.85	2.22	5.15	6.19
	c 10	24.9	16.5	5.1	B-2	7.68/8.63	2.15	4.13	4.75
	(N H )
	c 2	30.5	23.1	13.4	B-2	7.64/8.24	4.23	5.40	4.96
	c 4	28.7	24.0	10.9	B-1	8.79/9.09	4.14	5.43	5.42
	c 5	26.3	23.1	11.7	B-1	8.24/7.64	4.15	5.39	5.15
	c 7	28.7	27.2	15.7	B-1	7.41/7.89	2.66	3.18	4.02
	c 8	25.0	33.1	15.4	B-1	7.97/8.60	4.07	2.63	2.70
	c 9	28.8	25.9	11.8	B-1	8.99/9.01	3.45	5.84	6.53
	H )
	c 2	25.5	22.8	10.5	B-1	7.63/8.22	4.80	3.71	2.59
	(N H )
	c 1	27.9	28.6	3.2	B-1	7.34/7.90	2.67	2.92	4.15
	c 2	25.3	29.0	2.9	B-1	7.87/8.12	2.60	3.00	4.21
	H )
	c 2	27.8	28.5	6.8	B-1	7.53/8.11	2.63	2.92	4.21
	a The c 1

Table 2

 2 Bond lengths and energies for tetrahedral zinc-ligand complex models calculated by By single point calculation at QM/QM B3LYP/6-311+G(d,p):B3LYP/6-31G(d) level after optimization of the structures at B3LYP/6-31G(d,p) level; values between brackets are from single point calculations at QM/QM B3LYP/6-311+G(d,p):B3LYP/6-31G(d) level after optimization of the structures at B3LYP/6-31G level b Bond lengths in Zn-complexes optimized at B3LYP/6-31G(d,p) level; values between brackets are for Zncomplexes optimized at B3LYP/6-31G level

	DFT methods
		H N	His83 C Met382
		N	S	C
		H O	Zn	N	NH	MIO
		H				C
				His L		NH 2
				O		O	O HisL
	Model	Relative energies (kcal/mol) a	Zn-N His83	H	Distances (Å) b Zn-N HisL Zn-O HOH	Zn-S Met382
	1	1.00(0.44)	1.97(1.96)		2.02(2.01)	2.07(2.05)	2.42(2.50)
	2	0.00(0.00)	1.96(1.96)		2.03(2.01)	2.06(2.05)	2.42(2.51)
	3	2.32(1.57)	1.96(1.96)		2.02(2.01)	2.08(2.05)	2.41(2.49)
	4	1.84(0.19)	1.98(1.96)		2.03(2.02)	2.07(2.05)	2.44(2.51)

a

Table 3

 3 Mulliken atomic charges of the pro-S -hydrogens in zwitterionic -histidine, in zwitterionic -4-nitrohistidine, in a truncated N-MIO-intermediate model and in the tetrahedral Zn-complex models Calculations on the ligand conformations allowed within the HAL active site were performed at B3LYP/6-31G(d,p) level (values between brackets are from B3LYP/6-31G DFT calculations)

	Entry	Structure a	H S	H R
			0.176 (0.221)	0.133 (0.173)
		-Histidine	0.180 (0.222)	0.102 (0.166)
		-4-Nitrohistidine	0.199 (0.245)	0.135 (0.201)
	4	Zn-complex, Model 1	0.220(0.267)	0.172 (0.206)
	5	Zn-complex, Model 2	0.220 (0.265)	0.173 (0.208)
	6	Zn-complex, Model 3	0.220 (0.263)	0.174 (0.208)
	7	Zn-complex, Model 4	0.215 (0.261)	0.177 (0.210)

a

Acknowledgments

ALS thanks the financial support of Romanian National University Research Council (CNCSIS, PNII-TD-400). PL thanks the financial support by the Hungarian National Science Foundation (OTKA T-048854) and by the Hungarian National Office for Research and Technology (NKTH A2 2007 FLOWREAC).

Abbreviations

1GKM mod