Identification of functionally important residues in the pyridoxal-5'-phosphate-dependent catalytic antibody 15A9

Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.     

Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.     

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′‐phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP‐binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT‐pyridoxamine 5′‐phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300‐ to 500‐fold decrease in both the rate constant of L‐alanine half‐transamination and the k_cat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc.     

Controlling reaction specificity in pyridoxal phosphate enzymes

Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Crystal structures of aminotransferases Aro8 and Aro9 from Candida albicans and structural insights into their properties

Aminotransferases catalyze reversibly the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various aminotransferases acting on a range of substrates have been reported. Aromatic transaminases are able to catalyze the transamination reaction with both aromatic and acidic substrates. Two aminotransferases from C. albicans, Aro8p and Aro9p, have been identified recently, exhibiting different catalytic properties. To elucidate the multiple substrate recognition of the two enzymes we determined the crystal structures of an unliganded CaAro8p, a complex of CaAro8p with the PLP cofactor bound to a substrate, forming an external aldimine, CaAro9p with PLP in the form of internal aldimine, and CaAro9p with a mixture of ligands that have been interpreted as results of the enzymatic reaction. The crystal structures of both enzymes contains in the asymmetric unit a biologically relevant dimer of 55?kDa for CaAro8 and 59?kDa for CaAro9p protein subunits. The ability of the enzymes to process multiple substrates could be related to a feature of their architecture in which the active site resides on one subunit while the substrate-binding site is formed by a long loop extending from the other subunit of the dimeric molecule. The separation of the two functions to different chemical entities could facilitate the evolution of the substrate-binding part and allow it to be flexible without destabilizing the conservative catalytic mechanism.     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.     

Structural insights into catalysis by βC-S lyase from Streptococcus anginosus

Hydrogen sulfide (H₂S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H₂S production is associated with βC‐S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the α,β‐elimination of sulfur‐containing amino acids. When Lcd acts on L ‐cysteine, H₂S is produced along with pyruvate and ammonia. To understand the H₂S‐producing mechanism of Lcd in detail, we determined the crystal structures of substrate‐free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α‐aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP‐binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L ‐serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L ‐cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC‐S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC‐S lyases from oral bacteria. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis.Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.     

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.     

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.     

Pyridoxal 5'-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B₆ requiring enzymes. PLP reacts with apo-B₆ enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B₆ enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B₆ proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B₆ enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.     

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes.

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C alpha and C-4' positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular pKa of the monoprotic base. Thus the epsilon-ammonium group of the internal aldimine-forming lysine residue operates at C-4'-si-face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C alpha from the 4'-si-face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldimination processes and as a binding group for the alpha-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. -S-X-H-K-) with that for aspartate aminotransferase (e.g. -S-X-A-K-) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5'-phosphate: internal aldimine and stable L-aspartate external aldimine

The 1.8 ? resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5'-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and L-aspartate external aldimine forms are reported. The L-aspartate·deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with L-aspartate. This structure is compared to that of the α-methyl-L-aspartate·PLP external aldimine. Overlays with the corresponding pyridoxal 5'-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the L-aspartate·deazaPLP and α-methyl-L-aspartate·PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4'. In contrast, in the α-methyl-L-aspartate·PLP external aldimine, the ε-amino group of Lys258 is rotated ~70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.     

Lysine 238 is an essential residue for alpha,beta-elimination catalyzed by Treponema denticola cystalysin

Treponema denticola cystalysin is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the alpha,beta-elimination of l-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently approximately 50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 x 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the alpha,beta-elimination of l-cysteine and beta-chloro-l-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Calpha-proton from the substrate and possibly as a general acid protonating the beta-leaving group.     

Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.     

Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in a SAM-dependent iron-sulfur enzyme.     

Mapping of a hapten-binding site: molecular modeling and site-directed mutagenesis study of an anti-atrazine antibody

A three-dimensional model of the variable domain of the atrazine-specific Fab fragment K411B was constructed by molecular modeling using known structures of highly homologous immunoglobulins as templates. Molecular dynamic simulations and cross-reactivity data were used to predict residues responsible for the binding of the hapten 4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (iPr/Cl/C6) instead of atrazine. Specific binding pockets could be defined for the chlorine, the isopropylamino group and the C6-spacer of the hapten. The influence of various amino acids on hapten binding was investigated by site-directed mutagenesis, and the effect of these mutations was analyzed by capture ELISA using the hapten iPr/Cl/C6 and 4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (H/Cl/C6). GlyH100a seems to be important in determining the conformation of the heavy-chain complementarity determining region H3; replacing it with any other residue prevented the binding of the hapten. Altering residues responsible for the binding of the chlorine atom (TrpH33, GluH50 and TyrL96) decreased the affinity significantly. Hapten-spacer recognition can be attributed to the interaction with PheL32; replacing PheL32 by leucine reduced the affinity towards iPr/Cl/C6. A triple mutant Fab fragment (GlnL89Glu, ValH37Ile and GluL3Val) showed an affinity 5-fold greater towards iPr/Cl/C6 compared to the wild-type K411B, as a result of better recognition of the isopropylamino group of iPr/Cl/C6.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.     

Complexes of Cu2+ with pyridoxal-5'-phosphate and human and bovine serum albumin

The copper ion Cu2+ bound to serum albumin in the most strong center stabilizes aldimine bonds formed by PLP with epsilon-NH2 group of 4-Lys and alpha-NH2 1-Asp. The stoichometric ratio of the ternary albumin-PLP-Cu2+ complex is 1:2:1. The imidazole rings of histidine residues are involved in binding of copper ions in the first, second, third centers of the albumin molecule. In this case copper ions increase the binding of PLP with the protein stabilizing Schiff bases produced by epsilon-NH2 group of lysine and PLP. The cooper ion bound to serum albumin in the most strong center forms two types of complexes: with rhombic environment in neutral and alkaline media and axial one at pH less than 5,0. On formation of the ternary complex with PLP the rhombic environment is changed to axial.