Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for many enzymes involved in the metabolism of amino acids, amino acid derived metabolites and some amino sugars. PLP is one of the most versatile cofactors and the PLP-dependent enzymes catalyze a variety of reactions including transamination, decarboxylation, inter-conversion of L-and D-amino acids and removal or replacement of chemical groups bound at β or γ carbon of amino acids. The thesis describes the structural studies carried out on three PLP-dependent enzymes; N-acetylornithine aminotransferase (AcOAT), serine hydroxymethyltransferase (SHMT) and diaminopropionate ammonia lyase (DAPAL). Chapter 1 of the thesis begins with a brief introduction to PLP-dependent enzymes and their classification. This is followed by a review of structures of enzymes belonging to the subgroup II aminotransferases. The last section of chapter I contains a detailed description of the structures available till date for SHMT from various sources and the mutational studies carried out on SHMT. All the common experimental procedures and computational methods used for the current investigations are described in chapter II, as most of these are applicable to all structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization, and X-ray diffraction data collection. Computational methods include details of various programs used during data processing, structure determination, refinement, model building, structure validation and analysis. AcOAT is one of the key enzymes in arginine and lysine metabolism. AcOAT belongs to the fold type I (αfamily) subgroup II family of PLP dependent enzymes. Both S. typhimurium and E. coli have two genes each, one involved in the biosynthesis of arginine and another in the biodegradation of arginine. Biosynthetic AcOAT catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate and the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate in lysine biosynthesis. Meso-DAP and lysine, the products of lysine biosynthesis pathway, are known to function as cross-linking moieties in the peptidoglycan component of bacterial cell wall. Therefore N-acetylornithine aminotransferase could serve as a target for designing antibacterials. Chapter III gives the details of the work carried out on AcOAT. Two genes each from S. typhimurium and E. coli coding for biosynthetic and biodegradative AcOAT were cloned in E. coli, overexpressed and purified by Ni-NTA affinity chromatography. Of the four enzymes, biosynthetic AcOAT from S. typhimurium (sArgD) crystallized in the unliganded form and in the presence of the inhibitor gabaculine or one of the substrates L-glutamate, diffracted to a maximum resolution of 1.90 Å and contained a dimer in the asymmetric unit. The structure was determined by the molecular replacement method using human ornithine aminotransferase (hOAT) as the starting model. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop Lαk-βm in only one subunit. The Km and kcat/Km values determined with the purified sArgD suggested that the enzyme could accept both acetylornithine (AcOrn) and ornithine (Orn) as the substrates and had much higher affinity for AcOrn than for Orn. However, OAT accepts only Orn as the substrate. Comparison of the structurte of sArgD with T. thermophilus AcOAT and hOAT suggested that the higher specificity of sArgD towards AcOrn may not be due to specific differences in the active site residues but could result from minor conformational changes in some of them. sArgD was inhibited by gabaculine with an inhibition constant (Ki) of 7 µM and a second order rate constant (k2) of 0.16 mM-1s-1. The crystal structure of sArgD obtained in the presence of gabaculine and the spectral studies of sArgD with gabaculine suggested that the enzyme might have a low affinity for the PLP-gabaculine complex. Biosynthetic AcOAT from E. coli (eArgD) crystallized in the presence of gabaculine in hanging drop vapor diffusion method and diffracted X-rays only to a resolution of 3.5 Å. Two data sets were collected for the eArgD crystals. One of the data sets belonged to P1 (data 1) and the other to P321 space group (data 2) with a solvent content of ~70%. Data 1 was twinned and the unit cell was unusually large and could accommodate ~24 molecules in the asymmetric unit where as data 2 had four molecules in the asymmetric unit. Biodegradataive AcOAT from E. coli also crystallized in presence of gabaculine in hanging drop vapor diffusion method and suffered from low diffraction quality, where as that from S. typhimurium did not yield crystals. In chapter IV, X-ray crystallographic studies on various site specific mutants of SHMT from Bacillus stereotherophilus (bs) and a detailed comparison of structural data with the biochemical results in relation to mechanism of catalysis are presented. SHMT is a member of the α-class of PLP-dependent enzymes and catalyzes the reversible conversion of L-Ser and THF to glycine and 5,10-methylene THF. 5,10-methylene THF serves as a major source of one-carbon units in the biosynthesis of nucleotides and a few amino acids. SHMT also catalyses the cleavage of β-hydroxy amino acids like L-allo-threonine, transamination, racemization and decarboxylation reactions. SHMT shows increased activity along with enhanced nucleotide synthesis and therefore is a potential target for cancer chemotherapy. The availability of structural and biochemical data on SHMT from different sources ranging from human to E. coli enabled the identification of active site residues and a more critical examination of the role of these residues in the different steps of catalysis. The important mutants studied in the present investigation are E53Q, Y51F, Y61F, Y61A, Y60A, N341A and F351G of bsSHMT. The crystal structures of all these mutants are solved in the presence of various ligands, which gave many interesting results. E53, one of the active residues, interacts with the side chain hydroxyl group of serine bound to PLP in the wild type serine complex and N10 and formyl oxygen in the wild type glycine-FTHF complex. In E53Q glycine and serine complexes, glycine carboxyl and serine side chain were in two conformations, respectively, the new conformation being stabilized by their interaction with the mutated residue Q53. The structure of E53Q-Gly complex obtained in the presence and absence of 5-formyl THF(FTHF) showed an interesting case of enzyme memory in which the final conformational state depends on the way it was obtained and suggested that E53 is crucial for FTHF/THF binding. Though the spectrum showed that FTHF binds to the mutant initially, no density was observed for FTHF in the final structure. FTHF is believed to dissociate from the active site with prolonged incubation leaving behind a few significant conformational changes. Y51, one of the highly conserved tyrosines in SHMT, has hydrogen bonding interactions with the phosphate group of PLP and the active site lysine (K226) in bsSHMT. Mutation of Y51 to F resulted in significant changes at the active site. In all the structures of Y51F complexes, the phosphate group is in two conformations and F51 has moved away from the phosphate and in turn changed the position of Y61, another tyrosine in the active site. The residue Y61 is hydrogen bonded to R357 in the internal aldimine complex of bsSHMT. Addition of glycine/serine to bsSHMT resulted in the conformational change of Y61 away from R357 and towards E53, allowing the added glycine/serine to interact with R357. Mutation of Y61 to A did not bring significant structural changes. Structures of Y51F and Y61A mutants complexed with L-allo-Thr (cleaved to Gly by the wild type enzyme) showed that L-allo-Thr was not cleaved to glycine and acetaldehyde and confirmed the biochemical observation that these two residues are essential even for the THF-independent reaction. Residues Y60 and N341 are also highly conserved residues among SHMTs. Y60 stacks over PABA ring of FTHF in the wild type glycine-FTHF ternary complex. N341 has strong hydrogen bonding interactions with N1 and N8 atoms of the pteridine ring of FTHF. Mutation of either Y60 or N341 to A destroys the binding ability of FTHF/THF to the enzyme according to the biochemical and structural observations. The residue F351 exhibits different conformations in the two subunits of wild type glycine-FTHF ternary complex and is thought to be an important residue in determining the asymmetric binding of FTHF. Mutation of F351 to G did not affect the catalytic activity. Surprisingly, in the crystal structure obtained in the presence of L-allo-Thr, the ligand did not get cleaved to glycine, though in solution, the mutant is as active as the wild type enzyme. Chapter V describes the preliminary structural studies carried out on DAPAL from E. coli and S. typhimurium. DAPAL catalyzes the α, βelimination of both L-and D-diaminopropionate (DAP). DAP is the immediate precursor of two neurotoxins 3oxalyl and 2,3-dioxalyl DAP present in Lathyrus sativus, a grain legume rich in proteins and capable of growing well in drought conditions. The presence of these two neurotoxins precludes its use as a source of protein rich food. This enzyme is present only in bacteria and few species of actinomycetes. Unlike many other PLP-dependent enzymes, DAPAL does not catalyze any side reaction and is the only enzyme known to remove an amino group from the βcarbon of the substrate. The enzymes from E. coli (eDAPAL) and S. typhimurium (sDAPAL) produced diffraction quality crystals. However, crystals of sDAPAL did not survive heavy atom soaking and eDAPAL crystals suffered from poor reproducibility and severe non-isomorphism making it difficult to obtain suitable heavy atom derivatives for structure determination. Production of selenomethionine labelled proteins for these enzymes was initiated and thin crystals were obtained for eDAPAL. Improvement of the quality of these crystals is necessary in order to solve the structure of DAPAL by MAD method.

The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis. DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested. AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity. The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance. Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.