PyMod: sequence similarity searches, multiple sequence-structure alignments, and homology modeling within PyMOL.

BACKGROUND: In recent years, an exponential growing number of tools for protein sequence analysis, editing and modeling tasks have been put at the disposal of the scientific community. Despite the vast majority of these tools have been released as open source software, their deep learning curves often discourages even the most experienced users. RESULTS: A simple and intuitive interface, PyMod, between the popular molecular graphics system PyMOL and several other tools (i.e., [PSI-]BLAST, ClustalW, MUSCLE, CEalign and MODELLER) has been developed, to show how the integration of the individual steps required for homology modeling and sequence/structure analysis within the PyMOL framework can hugely simplify these tasks. Sequence similarity searches, multiple sequence and structural alignments generation and editing, and even the possibility to merge sequence and structure alignments have been implemented in PyMod, with the aim of creating a simple, yet powerful tool for sequence and structure analysis and building of homology models. CONCLUSIONS: PyMod represents a new tool for the analysis and the manipulation of protein sequences and structures. The ease of use, integration with many sequence retrieving and alignment tools and PyMOL, one of the most used molecular visualization system, are the key features of this tool.Source code, installation instructions, video tutorials and a user's guide are freely available at the URL http://schubert.bio.uniroma1.it/pymod/index.html.

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

In silico and in vitro validation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed.

Serine hydroxymethyltransferase (SHMT), a ubiquitous representative of the family of fold-type I, pyridoxal 5'-phosphate (PLP) dependent enzymes, catalyzes the reversible conversion of tetrahydrofolate (H4PteGlu) and serine to 5,10-CH2-H4PteGlu and glycine. Together with thymidylate synthase (TS) and dihydrofolate reductase (DHFR), SHMT participates to the thymidylate (dTMP) biosynthetic process. Elevated SHMT activity has been coupled to the increased demand for DNA synthesis in tumour cells. However, SHMT is the only enzyme of the thymidylate cycle yet to be targeted by chemotherapeutics. In this study, the interaction mode of SHMT with pemetrexed, an antifolate drug inhibiting several enzymes involved in folate-dependent biosynthetic pathways, was assessed. The mechanism of SHMT inhibition by pemetrexed was investigated in vitro using the human recombinant protein. The results of this study showed that pemetrexed competitively inhibits SHMT with respect to H4PteGlu with a measured Ki of 19.1±3.1 µM; this value was consistent with a Kd of 16.9±5.0 µM, measured by isothermal titration calorimetry. The binding mode of pemetrexed to SHMT was further investigated by molecular docking. The calculated interaction energy of pemetrexed in the active site of SHMT was -7.48 kcal/mol, and the corresponding predicted binding affinity was 36.3 µM, in good agreement with Kd and Ki values determined experimentally. The results thus provide insights into the mechanism of action of this antifolate drug and constitute the basis for the rational design of more selective inhibitors of SHMT.

Structural adaptation of serine hydroxymethyltransferase to low temperatures.

Structural adaptation of serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate dependent enzyme that catalyzes the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylene-tetrahydropteroylglutamate, synthesized by microorganisms adapted to low temperatures has been analyzed using a comparative approach. The variations of amino acid properties and frequencies among three temperature populations (psychrophilic, mesophilic, hyper- and thermophilic) of SHMT sequences have been tested. SHMTs display a general increase of polarity specially in the core, a more negatively charged surface, and enhanced flexibility. Subunit interface is more hydrophilic and less compact. Electrostatic potential of the tetrahydrofolate binding site has been compared. The enzyme from Psychromonas ingrahamii, the organism with the lowest adaptation temperatures, displayed the most positive potential. In general, the property variations show a coherent opposite trend in the hyperthermophilic population: in particular, increase of hydrophobicity, packing and decrease of flexibility was observed.

Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase--the role of evolutionarily conserved hydrophobic contacts.

According to their fold, pyridoxal 5'-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5'-phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of the quaternary structure.

Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5'-phosphate-dependent enzyme catalyzing CO(2) release from the alpha-carboxyl group of L-glutamate to yield gamma-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5'-phosphate-Lys(276) Schiff base with the distal nitrogen of His(465), the penultimate residue in the GadB sequence. Herein we show that His(465) has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His(465) contributes with n approximately 2.5 to the overall cooperativity of the system. The residual cooperativity (n approximately 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His(465), dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His(465), a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His(465) mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.

The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase.

Pyridoxal 5'-phosphate-dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site-directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydroxymethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis.

Structural adaptation of the subunit interface of oligomeric thermophilic and hyperthermophilic enzymes.

Enzymes from thermophilic and, particularly, from hyperthermophilic organisms are surprisingly stable. Understanding of the molecular origin of protein thermostability and thermoactivity attracted the interest of many scientist both for the perspective comprehension of the principles of protein structure and for the possible biotechnological applications through application of protein engineering. Comparative studies at sequence and structure levels were aimed at detecting significant differences of structural parameters related to protein stability between thermophilic and hyperhermophilic structures and their mesophilic homologs. Comparative studies were useful in the identification of a few recurrent themes which the evolution utilized in different combinations in different protein families. These studies were mostly carried out at the monomer level. However, maintenance of a proper quaternary structure is an essential prerequisite for a functional macromolecule. At the environmental temperatures experienced typically by hyper- and thermophiles, the subunit interactions mediated by the interface must be sufficiently stable. Our analysis was therefore aimed at the identification of the molecular strategies adopted by evolution to enhance interface thermostability of oligomeric enzymes. The variation of several structural properties related to protein stability were tested at the subunit interfaces of thermophilic and hyperthermophilic oligomers. The differences of the interface structural features observed between the hyperthermophilic and thermophilic enzymes were compared with the differences of the same properties calculated from pairwise comparisons of oligomeric mesophilic proteins contained in a reference dataset. The significance of the observed differences of structural properties was measured by a t-test. Ion pairs and hydrogen bonds do not vary significantly while hydrophobic contact area increases specially in hyperthermophilic interfaces. Interface compactness also appears to increase in the hyperthermophilic proteins. Variations of amino acid composition at the interfaces reflects the variation of the interface properties.

"Hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.

BACKGROUND: A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures. RESULTS: The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions. CONCLUSION: This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in the protein core and/or a better packing of alpha-helices with the rest of the structure, caused by the presence of hydrophobic beta-branched side chains.

Structural adaptation to low temperatures--analysis of the subunit interface of oligomeric psychrophilic enzymes.

Enzymes from psychrophiles show higher catalytic efficiency in the 0-20 degrees C temperature range and often lower thermostability in comparison with meso/thermophilic homologs. Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation. Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and by a lower activation energy requirement in comparison with meso/thermophilic counterparts. In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content. In this study, a comparative analysis of the structural characteristics of the interfaces between oligomeric psychrophilic enzyme subunits was carried out. Crystallographic structures of oligomeric psychrophilic enzymes, and their meso/thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank. The following structural parameters were calculated: overall and core interface area, characterization of polar/apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, internal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues. These properties were compared to those of meso/thermophilic enzymes. The results were analyzed using Student's t-test. The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface. Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface.

Pyridoxal 5'-phosphate enzymes as targets for therapeutic agents.

The vitamin B(6)-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.

The mechanism of addition of pyridoxal 5'-phosphate to Escherichia coli apo-serine hydroxymethyltransferase.

Previous studies suggest that the addition of pyridoxal 5'-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5'-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB.

Escherichia coli and other enterobacteria exploit the H+ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

CAMPO, SCR_FIND and CHC_FIND: a suite of web tools for computational structural biology.

The identification of evolutionarily conserved features of protein structures can provide insights into their functional and structural properties. Three methods have been developed and implemented as WWW tools, CAMPO, SCR_FIND and CHC_FIND, to analyze evolutionarily conserved residues (ECRs), structurally conserved regions (SCRs) and conserved hydrophobic contacts (CHCs) in protein families and superfamilies, on the basis of their 3D structures and the homologous sequences available. The programs identify protein segments that conserve a similar main-chain conformation, compute residue-to-residue hydrophobic contacts involving only apolar atoms common to all the 3D structures analyzed and allow the identification of conserved amino-acid sites among protein structures and their homologous sequences. The programs also allow the visualization of SCRs, CHCs and ECRs directly on the superposed structures and their multiple structural and sequence alignments. Tools and tutorials explaining their usage are available at http://schubert.bio.uniroma1.it/SCR_FIND, http://schubert.bio.uniroma1.it/CHC_FIND and http://schubert.bio.uniroma1.it/CAMPO.

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5'-phosphate-dependent enzymes.

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction.

Structures of gamma-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5'-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with gamma-ethynyl-GABA and with the antiepilepsy drug vigabatrin.

Gamma-aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, gamma-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-A resolution) and in complex with vigabatrin as well as with the close analogue gamma-ethynyl-GABA (to 2.3 and 2.8 A, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5'-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases.

Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase.

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.

Catalytic and thermodynamic properties of tetrahydromethanopterin-dependent serine hydroxymethyltransferase from Methanococcus jannaschii.

The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of Cbeta of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability.

Threonine aldolase and alanine racemase: novel examples of convergent evolution in the superfamily of vitamin B6-dependent enzymes.

Vitamin B(6)-dependent enzymes may be grouped into five evolutionarily unrelated families, each having a different fold. Within fold type I enzymes, L-threonine aldolase (L-TA) and fungal alanine racemase (AlaRac) belong to a subgroup of structurally and mechanistically closely related proteins, which specialised during evolution to perform different functions. In a previous study, a comparison of the catalytic properties and active site structures of these enzymes suggested that they have a catalytic apparatus with the same basic features. Recently, recombinant D-threonine aldolases (D-TAs) from two bacterial organisms have been characterised, their predicted amino acid sequences showing no significant similarities to any of the known B(6) enzymes. In the present work, a comparative structural analysis suggests that D-TA has an alpha/beta barrel fold and therefore is a fold type III B(6) enzyme, as eukaryotic ornithine decarboxylase (ODC) and bacterial AlaRac. The presence of both TA and AlaRac in two distinct evolutionary unrelated families represents a novel and interesting example of convergent evolution. The independent emergence of the same catalytic properties in families characterised by completely different folds may have not been determined by chance, but by the similar structural features required to catalyse pyridoxal phosphate-dependent aldolase and racemase reactions.