Gotowa bibliografia na temat „Propionate Kinase (TdcD)”

Acetate and propionate are low molecular mass carbon compounds found abundantly in the soil. Although these compounds have been extensively used as food preservatives because of their ability to inhibit microbial growth, surprisingly, bacteria such as Escherichia coli and Salmonella typhimurium are able to grow on propionate as their sole carbon and energy source. Only in the presence of glucose, acetate and other short chain fatty acids inhibit microbial growth. Propionate is produced during the β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates, oxidative degradation of branched-chain amino acids such as valine and isoleucine. They are also produced during the catabolism of threonine, methionine, thymine and cholesterol. In Escherichia coli and Salmonella typhimurium, enzymes involved in the degradation of L-serine and L-threonine to acetate and propionate, respectively, are encoded by the anaerobically regulated tdc operon. L-threonine is anaerobically degraded to propionate in four consecutive reaction steps catalyzed by biodegradative threonine deaminase (TdcB), 2-ketobutyrate formate lyase (TdcE), phosphotransacetylase (Pta) and propionate kinase (TdcD). Detailed studies on the structure and function of two of these enzymes have earlier been carried out in our laboratory. However, these studies did not reveal the precise substrate binding site in Salmonella typhimurium TdcD (StTdcD). It was also not possible to provide a satisfactory explanation of the structural basis of substrate specificity. The present studies were therefore aimed at locating the substrate binding site, elucidating the structural basis of substrate specificity and mechanism of catalysis of StTdcD. Oxalic acid is toxic to almost all organisms and its excessive occurrence leads to a variety of pathological conditions. In humans and other vertebrates, secretion of oxalic acid leads to formation of low soluble calcium oxalate, which precipitates as kidney stones. Formation of kidney stones is aggravated by lack of enzymes that catabolize oxalate. Oxalate oxidase, oxalate decarboxylase and oxalyl-CoA decarboxylase constitute three distinct categories of oxalate degrading enzymes. Photorhabdus luminescens is a Gram-negative, symbiotic bacterium associated with the entomopathogenic nematodes of the family Heterorhabditidae. Novel insecticidal genes from these symbiotic bacteria are now being examined for their potential in generating pest resistant transgenic plants. As part of this project, the three-dimensional X-ray crystal structure of an oxalate oxidase (OXDC) enzyme from Photorhabdus luminescens (PlOXDC) was determined. The introductory chapter (Chapter I) of the thesis presents the earlier investigations carried out in the laboratory on the structure and function of StTdcD. It also provides a summary of the earlier literature pertaining to propionate metabolism in S. typhimurium. The crystal structure of StTdcD in the apo form as well as in complex with ADP and the non-hydrolysable nucleotide analog AMPPNP were determined by earlier Dr. Simanshu ( Simanshu et al., 2005 , 2008 ). Subsequently, Dr Chittori determined the structures of the enzyme in complex with various other nucleotides ( Chittori et al., 2013 ). These studies along with enzyme assays performed by Chittori revealed that StTdcD possesses broad specificity and it could be activated by various nucleotides and metal ions and catalyzes phosphorylation of both propionate and acetate ( Chittori et al., 2013 ). In spite of these extensive studies, the precise mode of binding of the substrate propionate to StTdcD could not be elucidated. The chapter also presents a summary of the literature on oxalate, its toxic effects and enzymes that degrade oxalate. The importance of structural and functional studies on oxalate degrading enzymes and other enzymes encoded by Photorhabdus luminescens is also briefly discussed. All the experimental protocols and computational methods applicable for most of the investigations reported in Chapters 4, 5 and 6 are presented in Chapter II. The experimental procedures described include cloning, overexpression, purification, enzymatic assays, crystallization and X-ray diffraction data collection. Computational methods covered include summary of crystallographic theory and details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. The databases that were used in the course of these investigations are also cited. The experience gained during attempts to determine the structure of StTdcD by single wavelength anomalous dispersion (SAD) method is described in Chapter III. The impetus for this work was the urge to examine the power of SAD technique making use of a newly acquired rotating anode X-ray generator equipped with a chromium anode. As expected, the structure determined by SAD was very close to the earlier determined structure of StTdcD. The structure contained a citrate, which was part of the crystallization cocktail at the active site. This is in contrast with acetate kinase, where it was found that citrate binds at the dimeric interface. The present studies demonstrated that the identification of a plausible regulatory site at the interface of dimeric structure in acetokinases based on the structure of acetate kinase (Chittori et al., 2013) is not valid for propionate kinase. Extensive efforts carried out to obtain structures of StTdcD and its mutants StTdcD A88V and StTdcD G207A complexed with either the substrate or substrate analogues provided several crystal structures. In most of these structures, the ligand was bound at a position distinct from the substrate binding site. These structures and their analysis are described Chapter IV. Asn206 was transformed from a disallowed region to an allowed region of the Ramachandran map in these structures whenever an anion was bound at the position corresponding to the γ- phosphate of the nucleotide substrate. This structural transformation might enhance the affinity of the enzyme for the substrate. In the structure of StTdcD A88V in complex with AMPPNP, AMPPNP was found to be cleaved to AMP and PNP either due to catalytic activity of the enzyme or due to radiation damage. The released PNP probably reacted with propionate forming propionyl-pyrophosphate. These structures also demonstrate that the nucleotide site readily accommodates the substrate or substrate analogues in the absence of a bound nucleotide. StTdcD catalyzes the Mg2+ ion dependent inter-conversion of propionate and ATP to propionyl phosphate and ADP. Two distinct catalytic mechanisms have been proposed for the phosphoryl transfer reaction catalyzed by acetokinase family enzymes: 1) direct-in-line transfer mechanism and 2) triple displacement mechanism (Anthony and Spector, 1972; Matte et al., 1998). In both, the configuration of the transferred phosphate undergoes an inversion, which has been experimentally demonstrated. Structural studies carried out with the view of elucidating the catalytic mechanism of StTdcD is described in Chapter V. Fortunately, it was possible to obtain the crystal structures of StTdcD and its mutants with propionate and AMPPNP bound at the active site. The structure supported an associative SN2 type direct in-line transfer mechanism of catalysis. The studies also revealed that Arg236 and His175 are catalytically important residues. As suggested earlier, Ala88 has a major role in specificity determination. However, Ala88 is not the sole determinant of specificity. Active site volume determining residues, Arg86, His118, Asp143 and the segment Pro116-Leu117-His118 are also important for substrate specificity. The catalytic mechanism proposed in this chapter may also be applicable to other acetokinase family members. The final Chapter VI describes three different crystal structures of PlOXDC. As expected from sequence similarity with B. subtilis and T. maritima OXDCs, PlOXDC polypeptide was found to possess a bicupin structure. However, the functional unit was a trimer in contrast to BsOXDC which functions as a hexamer. The difference is shown to be due to the disorder in the amino terminal segment of PlOXDC. The polypeptide was truncated during purification by a non-specific cleavage at residue Lys26 either by thrombin used for cleaving the covalently attached GST tag or by some other protease. However, in the crystal structure, the amino terminal 90 residues were disordered. The observed trimeric form of PlOXDC may represent its inherent nature or a result of the missing N-terminal residues. There is some controversy in the literature on whether both or only one cupin domain of the protomer is catalytically active. The structures presented in this chapter provided significant information on the mode of ligand binding to PlOXDC. In one of the structures, EDO was bound to both the cupin domains and was involved in similar interactions with protein atoms. This may imply that the substrate binds at both the sites and both cupin domains may have catalytic function. The thesis ends with a short note on future perspectives. It is clear that substantial work has been carried out on acetokinases. These studies have provided significant understanding of their structure and function. In the future, appropriate site-specific mutations of the substrate specificity determining residues may be made and their effect on enzyme specificity could be studied. Similarly, mutagenesis experiments could be performed to inter-convert acetate, propionate and butyrate kinases. These studies will provide deeper insights on intricacies of enzyme function. In contrast to the work on short chain fatty acid kinases, work on OXDC should be considered preliminary and further biochemical and structural studies are needed to illustrate the catalytic mechanism and examine if the protein is a suitable candidate for generating transgenic crops resistant to insect pests. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis.

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.
List of Publications with Abstract 1. Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms. Chittori S, Savithri HS, Murthy MR. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Dec 1;67(Pt 12):1658-61. Acetate kinase (AckA) catalyzes the reversible transfer of a phosphate group from acetyl phosphate to ADP, generating acetate and ATP, and plays a central role in carbon metabolism. In the present work, the gene corresponding to AckA from Salmonella typhimurium (StAckA) was cloned in the IPTG-inducible pRSET C vector, resulting in the attachment of a hexahistidine tag to the N-terminus of the expressed enzyme. The recombinant protein was overexpressed, purified and crystallized in two different crystal forms using the microbatch-under-oil method. Form I crystals diffracted to 2.70 Å resolution when examined using X-rays from a rotating-anode X-ray generator and belonged to the monoclinic space group C2, with unit-cell parameters a = 283.16, b = 62.17, c = 91.69 Å, β =93.57°. Form II crystals, which diffracted to a higher resolution of 2.35 Å on the rotating-anode X-ray generator and to 1.90 Å on beamline BM14 of the ESRF, Grenoble, also belonged to space group C2 but with smaller unit-cell parameters (a = 151.01, b = 78.50, c = 97.48 Å, β = 116.37°). Calculation of Matthews coefficients for the two crystal forms suggested the presence of four and two protomers of StAckA in the asymmetric units of forms I and II, respectively. Initial phases for the form I diffraction data were obtained by molecular replacement using the coordinates of Thermotoga maritima AckA (TmAckA) as the search model. The form II structure was phased using a monomer of form I as the phasing model. Inspection of the initial electron-density maps suggests dramatic conformational differences between residues 230 and 300 of the two crystal forms and warrants further investigation. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/22139191 2. Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity. Chittori S, Savithri HS, Murthy MR. J Struct Biol. 2011 Apr;174(1):58-68. 2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/20970504 3. Preliminary X-ray crystallographic analysis of 2-methylcitrate synthase from Salmonella typhimurium. Chittori S, Simanshu DK, Savithri HS, Murthy MR. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Apr 1;66(Pt 4):467-70. Analysis of the genomic sequences of Escherichia coli and Salmonella typhimurium has revealed the presence of several homologues of the well studied citrate synthase (CS). One of these homologues has been shown to code for 2-methylcitrate synthase (2-MCS) activity. 2-MCS catalyzes one of the steps in the 2-methylcitric acid cycle found in these organisms for the degradation of propionate to pyruvate and succinate. In the present work, the gene coding for 2-MCS from S. typhimurium (StPrpC) was cloned in pRSET-C vector and overexpressed in E. coli. The protein was purified to homogeneity using Ni-NTA affinity chromatography. The purified protein was crystallized using the microbatch-under-oil method. The StPrpC crystals diffracted X-rays to 2.4 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 92.068, b = 118.159, c = 120.659 A, alpha = 60.84, beta = 67.77, gamma = 81.92 degrees . Computation of rotation functions using the X-ray diffraction data shows that the protein is likely to be a decamer of identical subunits, unlike CSs, which are dimers or hexamers. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/20383024 4. Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate. Simanshu DK, Chittori S, Savithri HS, Murthy MR. J Biosci. 2007 Sep;32(6):1195-206. In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/17954980 5. Structure of the putative mutarotase YeaD from Salmonella typhimurium: structural comparison with galactose mutarotases. Chittori S, Simanshu DK, Savithri HS, Murthy MR. Acta Crystallogr D Biol Crystallogr. 2007 Feb;63(Pt 2):197-205. Salmonella typhimurium YeaD (stYeaD), annotated as a putative aldose 1-epimerase, has a very low sequence identity to other well characterized mutarotases. Sequence analysis suggested that the catalytic residues and a few of the substrate-binding residues of galactose mutarotases (GalMs) are conserved in stYeaD. Determination of the crystal structure of stYeaD in an orthorhombic form at 1.9 A resolution and in a monoclinic form at 2.5 A resolution revealed this protein to adopt the beta-sandwich fold similar to GalMs. Structural comparison of stYeaD with GalMs has permitted the identification of residues involved in catalysis and substrate binding. In spite of the similar fold and conservation of catalytic residues, minor but significant differences were observed in the substrate-binding pocket. These analyses pointed out the possible role of Arg74 and Arg99, found only in YeaD-like proteins, in ligand anchoring and suggested that the specificity of stYeaD may be distinct from those of GalMs. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/17242513 6. Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium. Simanshu DK, Chittori S, Savithri HS, Murthy MR. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt 3):275-8. Biodegradative threonine deaminase (TdcB) catalyzes the deamination of L-threonine to alpha-ketobutyrate, the first reaction in the anaerobic breakdown of L-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to L-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni-NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 A, alpha = 103.09, beta = 94.70, gamma = 112.94 degrees) and II (a = 56.68, b = 76.83, c = 78.50 A, alpha = 66.12, beta = 89.16, gamma = 77.08 degrees) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 A (crystal form I) and 1.7 A (crystal form II) were collected at 100 K using an in-house X-ray source. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/16511321 7. Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase. Anindya R, Chittori S, Savithri HS. Virology. 2005 Jun 5;336(2):154-62. Pepper vein banding virus (PVBV), a member of the genus potyvirus, is a single-stranded positive-sense RNA virus and it primarily infects plants of the family Solanaceae. Genome organization and gene expression strategy of the potyviruses are similar to the picornaviruses, although they infect widely different hosts and have distinctly different morphologies. The genomic RNA of PVBV has a viral genome-linked protein (VPg) at the 5'-terminus and a poly(A) tail at the 3'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV NIb and VPg were over-expressed in Escherichia coli and purified under non-denaturing conditions. PVBV NIb was found to be active as polymerase and it could uridylylate the VPg in a template independent manner. N- and C-terminal deletion analysis of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation. The amino acid residue uridylylated by PVBV NIb was identified to be Tyr 66 by site-directed mutagenesis. It is possible that in potyviruses, replication begins with uridylylation of VPg which acts as primer for progeny RNA synthesis. Link for the complete article: http://www.ncbi.nlm.nih.gov/pubmed/15892957

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.