32,102 research outputs found
Structural and Functional Analysis of Proteins involved in Microbial Stress Tolerance and Virulence
No full textThe genus Salmonella consists of pathogenic gram negative organisms which infect intestines of birds, animals and humans. They are the causative agents of salmonellosis which is characterised by diarrhoea, nausea, fever and abdominal cramps. If not treated in time, salmonellosis can also be fatal. Salmonella genus is divided into two species Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies out of which the subspecies enterica has many of the pathogenic serovars of this species. Salmonella typhimurium is a server in the subspecies enterica of Salmonella enterica species.
Transmission of salmonellosis takes place through contaminated food and water. When the organism enters a host, it encounters a range of hostile environments such as acidic pH, lack of oxygen as well as immune response of the host. In order to establish infection, the bacterium needs to survive under stressful conditions and propagate itself. Various proteins are induced in cells under unfavourable conditions that protect them in such situations. One such group of proteins belongs to the Universal Stress Protein (USP) family.
Universal Stress Proteins are a set of proteins induced in organisms when it is exposed to a variety of environmental insults including heat shock, nutrient starvation, presence of toxic compounds, etc. Although survival in adverse conditions is mediated by induction of this group of proteins, the precise mechanism of cellular protection has not been elucidated yet. The functional role of a protein is directly related to its three-dimensional structure and hence important insights can be gained regarding the role of these proteins by determining their structures. The structures of two Universal Stress Proteins from S. typhimurium; a single domain protein, YnaF and another tandem USP domain protein, YdaA were determined by X-ray crystallography and biochemical analysis was carried out on them. Guided by structure, plausible roles for both the proteins in stress tolerance of S. typhimurium have been proposed.
Additionally, work was also carried out on phosphomannose isomerise from S. typhimurium. Phosphomannose isomerase is a housekeeping enzyme which catalyses the interconversion of mannose-6-phosphate and fructose-6-phosphate. Mannose is important for mannosylation of various lipids and proteins which form an important component of bacterial and fungal cell walls. Presence of a functional phosphomannose isomerise enzyme is important as it helps the organism survive adverse conditions by forming a strong cell wall which shields it from harmful environments. Moreover, phosphomannose isomerase was also found to be essential for virulence of Leishmania mexicana and Cryptococcus neoformans. The structure of phosphomannose isomerase from S. typhimurium was determined in our laboratory in the year 2009. However, in the earlier studies, the catalytically important residues had not been identified and mechanism of isomerisation was not established. Structural analysis, site directed mutagenesis and biochemical assays were used to identify key residues in the active site of StPMI. Identification of these residues might help in deciphering the catalytic mechanism which will eventually be useful to develop inhibitors that arrest the growth of Salmonella as well as other microorganisms.
The work reported in this thesis describes the efforts made to enhance our understanding of functional aspects of the two Universal Stress Proteins, YnaF and YdaA and phosphomannose isomerase from S. typhimurium.
Chapter 1 begins with a brief introduction to the kinds of unfavourable environments encountered by microorganisms and their strategies of adaptation. This is followed by a review of the literature on Universal Stress Proteins, which are induced in many organisms in response to arrest of or perturbations in the growth rate. Structural, biochemical and evolutionary aspects of members of the family have also been discussed. Subsequently, a brief description of the earlier work carried out on another enzyme important in stress tolerance, phosphomannose isomerase, has been documented. A detailed account of mechanisms of isomerisation carried out by aldose ketose isomerases and identification of important strategies for determination of mechanism of phosphomannose isomerase catalysed reaction have then been provided. The chapter ends with a summary of aims and objectives of the present work.
Chapter 2 describes the various experimental techniques and computational methods used during the course of this thesis work. Isolation of plasmids, overexpression and purification of protein, site directed mutagenesis, biochemical assays, crystallisation of proteins, X ray diffraction data collection form a part of the experimental aspect and have been described in detail. Brief descriptions of the programs used and principles behind computational methods used for structure determination (including data processing, phasing, model building and refinement), validation and analysis have also been provided.
Chapter 3 includes the structural and functional studies carried out on YdaA, a tandem USP domain protein from S. typhimurium. Expression, purification, crystallisation and structure determination of YdaA in its native and ADP bound forms are described in the chapter. Biochemical assays with radiolabelled ATP showed that YdaA was an ATPase. The crystal structure of YdaA complexed with ATP revealed the presence of ADP (hydrolysis product of ATP) only in the C-terminal domain of the protein. Based on structural analysis and presence of ATP binding motif in the C-terminal domain, it could be hypothesized that ATP hydrolysis activity of the protein is confined to the C-terminal domain of the protein. The N-terminal domain of the protein was found to play another interesting role. A zinc binding site could be identified in the N terminal domain based on structural analysis and elemental X-ray absorption studies done at the synchrotron. Site directed mutagenesis and biochemical experiments suggested that zinc binding in the N-terminal domain was not related to ATPase activity of the C-terminal domain. Additionally, an intermediate of lipid A biosynthesis pathway UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetyl glucosamine was found bound to the N-terminal domain of YdaA. Lipid A is the membrane anchor of polysaccharides in the outer membrane of gram negative organisms and the intermediate occurs at the committed step of the pathway. However, no similarities could be identified between YdaA and members of the relevant biosynthetic pathway. Therefore, YdaA is unlikely to play a catalytic role in the same pathway but can function as a carrier molecule. A plausible link between the N- and C-terminal domains of YdaA could be identified by structural analysis. Many catalytically suitable residues from the N-terminal domain were found to be close to the β-phosphate of ADP bound to the C-terminal domain. Hence YdaA was identified to be a zinc binding ATPase which might play some yet unidentified role in lipid A biosynthesis pathway.
Chapter 4 describes the attempts made towards understanding the functional role of YnaF, a single domain USP from S. typhimurium. A description of the expression, purification, crystallisation and X ray diffraction techniques used for structure determination of YnaF and its single site mutant have been provided in detail. Gel filtration, dynamic light scattering studies and the crystal structure determination of YnaF showed a tetrameric organisation of four USP protomers stabilised in the centre by chloride ions. Additionally, YnaF crystallised with a bound ATP even though ATP was not included in the crystallisation cocktail. Biochemical assays on YnaF with radiolabelled ATP showed that it was inactive with respect to ATP hydrolysis. When selected mutations that disrupt chloride binding were made, YnaF was converted to an active ATPase. The crystal structure of the mutant complexed with an ATP analogue revealed key differences at the active site in comparison with that of the wild type and allowed identification of residues that might be important for ATP hydrolysis in this group of proteins. Hence YnaF might play the role of a sensor protein in some signal transduction pathway involving chloride ions in bacteria. A structure based analysis and comparison of USPs from the Protein Data Bank with the structures of YnaF and YdaA is summarised at the end of this chapter.
Chapter 5 describes the efforts carried out towards determination of mechanism of isomerisation catalysed by phosphomannose isomerise (PMI). Earlier reports suggest that the enzyme catalyses the reversible isomerisation of mannose-6-phosphate and fructose-6-phosphate via formation of a cis-enediol intermediate. The structure of phosphomannose isomerase from S. typhimurium has been reported by our laboratory. The enzyme is a monomer with three domains; a catalytic domain, a carboxy terminal domain and an α-helical domain. Residues from the catalytic domain were found to coordinate a zinc ion. Overexpression, purification, co crystallisation experiments and soaking studies carried out on crystals of PMI and its single site mutants are outlined in this chapter. The structure of a complex of PMI with mannose-6-phosphate at pH 7.0 revealed the presence of a blob of density close to the zinc binding site which was confirmed to be the active site by analysis of conservation of residues in the site. Based on site directed mutagenesis, activity studies and analysis of structure of PMI, zinc was identified to play an important role in maintaining the structural integrity of the active site. Electrostatic surface analysis of the structure of PMI revealed that the zinc ion might also play the role of anchoring phosphate moiety of the substrate in a highly negatively charged active site pocket. Activity assays following site directed mutagenesis studies eliminated the role of Glu264 in catalysis and implicated two lysines, Lys86 and Lys132 as the possible base in the reaction. The plausible role of a highly conserved residue Arg274 was also proposed based on comparison of structures of wild type and mutant PMIs.
The future prospects of the work are briefly discussed towards the end of the thesis. Further experiments and analysis required to obtain better understanding of the functions of these proteins have been discussed.
The Appendix section describes extensive crystallisation attempts that were carried out on the enzyme sorbitol-6-phosphate-dehydrogenase from S. typhimurium which catalyses the isomerisation reaction between sorbitol-6-phosphate and glucose-6-phosphate using NADPH as the cofactor. Needle shaped crystals were obtained which diffracted to a poor resolution of 7-8 Å at our in house X ray facility. Attempts to improve the quality of the crystals like co crystallisation with substrate and its analogues, soaking in various compounds and seeding are briefly described.
The following manuscripts based on work described in this thesis have been published or will be communicated for publication.
1. Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.
Bangera M., Panigrahi R., Sagurthi S.R., Savithri H.S., Murthy M.R.N.
Journal of Structural Biology, 2015 Mar; 189 (3): 238-50.
2. Structural and functional insights into phosphomannose isomerise: role of zinc and catalytic residues.
Bangera M., Savithri H.S., Murthy M.R.N.
Manuscript under preparatio
Structural Studies On Enzymes From Salmonella Typhimurium Involved In Propionate Metabolism: Biodegradative Threonine Deaminase, Propionate Kinase And 2-Methylisocitrate Lyase
No full textI formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics
Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an
attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems.
By this time (beginning of 2002), I had finished my first semester and a major
part of the course work, so we decided to start a new project focusing on some of the
unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his
Ph. D. from the lab, helped me in literature work and in finding targets for structural
studies. Finally, we decided to target enzymes involved in the propionate etabolism.
The pathways for propionate metabolism in Escherichia coli as well as Salmonella
typhimurium were just established and there were no structural information available for
most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and
succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle.
Most of the enzymes involved in 2-methylcitrate pathway were not characterized
biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize
these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected
a complete diffraction data to a resolution of 2.5 Å which was later on extended to a
resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems.
Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the
plausible rationale for different substrate specificities of these two enzymes. Due to
unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good
diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we
decided to target other enzymes involved in propionate metabolism.
We looked into the literature for the metabolic pathways by which propionate is
synthesized in the Salmonella typhimurium and finally decided to target enzymes present
in the metabolic pathway which converts L-threonine to propionate. Formation of
propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme
towards propionate than acetate.
One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron
density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies
suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments.
By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally
successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme.
The structural studies carried out on these three enzymes have provided structural
as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future
Experimental investigation into the effect of substrate clamping on the piezoelectric behaviour of thick-film PZT elements
No full textThis paper details an experimental investigation of the clamping effect associated with thick-film piezoelectric elements printed on a substrate. The clamping effect reduces the measured piezoelectric coefficient, d33, of the film. This reduction is due to the influence of the d31 component in the film when a deformation of the structure occurs, by either the direct or indirect piezoelectric effect. Theoretical analysis shows a reduction in the measured d33 of 62%, i.e. a standard bulk lead zirconate titanate (PZT)-5H sample with a manufacturer specified d33 of 593pC/N would fall to 227.8pC/N. To confirm this effect, the d33 coefficients of five thin bulk PZT-5H samples of 220µm thickness were measured before and after their attachment to a metallized 96% alumina substrate. The experimental results show a reduction in d33 of 74% from 529pC/N to 139pC/N. The theoretical analysis was then applied to existing University of Southampton thick-film devices. It is estimated that the measured d33 value of 131pC/N of the thick-film devices is the equivalent of an unconstrained d33 of 345pC/N
Structural Studies on SeMV Chimeras and TSV : Insights into Capsid Assembly
No full textAssembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV).
CP of SeMV, a T=3 plant virus, consists of a disordered N-terminal R-domain and an ordered S-domain. The importance of the R-domain in the assembly was probed by replacement with polypeptides such as the B-domain of Staphylococcus aureus protein A and polypeptides P10 and P8 of SeMV. These chimera assembled into T=3 or larger virus like particles (VLPs). Addition of divalent cations resulted in the formation of heterogeneous nucleoprotein complexes that disappeared upon treatment with EDTA/RNAse. One of the chimeras (N∆65-B) purified in a dimeric form by affinity chromatography assembled into T=1 VLPs during crystallization. The three dimensional structure of these VLPs showed that they were devoid of divalent ions and the B-domain was disordered. These studies demonstrate the importance of N-terminal residues, metal ions in virus assembly and robustness of the assembly process. Also, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications.
Tobacco streak virus (TSV) is a polymorphic virus and a major plant pathogen. TSV capsids encapsidate the tri-partite ss-RNA genome of the virus in three spheroidal particles of diameters 27, 30 and 33 nm, respectively. CPs of ilarviruses are also involved in genome activation. The labile nature of ilarviruses has posed difficulties in their structure determination. This thesis describes the first crystal structure of truncated TSV-CP. The core of TSV CP conforms to the canonical β-barrel jelly roll tertiary structure found in other viral coat proteins. Dimers of CP with swapped C-terminal arms (C-arm) were observed in the two crystal structures determined. The C-arm was found to be flexible and responsible for the polymorphic and pleomorphic nature of TSV capsids. Mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was also found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses
Metabolic Adaptation For Utilization Of Short-Chain Fatty Acids In Salmonella Typhimurium : Structural And Functional Studies On 2-methylcitrate Synthase, Acetate And Propionate Kinases
No full textThree-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 pos
A 2 h periodic variation in the low-mass X-ray binary Ser X-1
No full textSpectroscopy of the low-mass X-ray binary Ser X-1 using the Gran Telescopio Canarias have revealed a ?2 h periodic variability that is present in the three strongest emission lines. We tentatively interpret this variability as due to orbital motion, making it the first indication of the orbital period of Ser X-1. Together with the fact that the emission lines are remarkably narrow, but still resolved, we show that a main-sequence K dwarf together with a canonical 1.4 M? neutron star gives a good description of the system. In this scenario, the most likely place for the emission lines to arise is the accretion disc, instead of a localized region in the binary (such as the irradiated surface or the stream-impact point), and their narrowness is due instead to the low inclination (?10°) of Ser X-1
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