Interaction of Sesbania Mosaic Virus Movement Protein with VPg and P10: Implication to Specificity of Genome Recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 5′ end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement

Mutation of interfacial residues disrupts subunit folding and particle assembly of Physalis mottle tymovirus

Virus-like particles (VLPs) serve as excellent model systems to identify the pathways of virus assembly. To gain insights into the assembly mechanisms of the Physalis mottle tymovirus (PhMV), six interfacial residues, identified based on the crystal structure of the native and recombinant capsids, were targeted for mutagenesis. The Q37E, Y67A, R68Q, D83A, I123A, and S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble. However, except for the S145A mutant, which assembled into VLPs similar to that of wild type rCP capsids, all the other mutants failed to assemble into VLPs. Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies. The rCP mutants locked into such conformations probably lack the structural signals/features that would allow them to assemble into capsids. Thus, the mutation of residues involved in inter-subunit interactions in PhMV disrupts both subunit folding and particle assembly

2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger: a novel decarboxylase

2,3-Dihydroxybenzoic acid decarboxylase, the last enzyme in the fungal metabolism of indole to catechol, catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol. Unlike most other decarboxylases, this enzyme does not require a cofactor, underlining the importance of active-site residues in the reaction mechanism. Earlier studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidyanathan, C. S. (1989) Biochem. Biophys. Res. Commun. 165, 20-261, have shown that the sulfhydryl agent N-ethylmaleimide (MalNEt) inactivated the enzyme by modifying a single class of cysteine residues and that this inactivation was prevented in the presence of salicylate, a substrate analogue. In the present study, this essential cysteine residue has been identified by specific labelling with [14C]- MalNEt using the differential labelling technique. The stoichiometry of incorporation of [I4C]MalNEt was approximately one/subunit of the homotetrameric protein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequent reverse-phase chromatography of the peptide mixture. The sequence of the major radioactive peptide that was identified to be the active-site peptide, was LLGLAETCK. A search for sequences similar to this active-site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constructed from the sequences of peptides derived from enzymic cleavage of the protein using endoproteinase Glu-C and trypsin did not share any significant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first report on the active-site peptide and the partial primary structure of a non-oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus

Studies on plant aspartate transcarbamylase purification and properties of the enzyme from mung-bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources

cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/1 bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5′-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5′-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55°C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1-K6)-SHMT (55°C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8 ± 0.1 M) and des-(A1-K6)-SHMT (1.7 ± 0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5′-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT

NSs Encoded by Groundnut Bud Necrosis Virus Is a Bifunctional Enzyme

Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus

Serine hydroxymethyltransferase (SHMT), a member of the α-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage

Functional Characterization of Pepper Vein Banding Virus-Encoded Proteins and Their Interactions: Implications in Potyvirus Infection

Pepper vein banding virus (PVBV) is a distinct species in the Potyvirus genus which infects economically important plants in several parts of India. Like other potyviruses, PVBV encodes multifunctional proteins, with several interaction partners, having implications at different stages of the potyviral infection. In this review, we summarize the functional characterization of different PVBV-encoded proteins with an emphasis on their interaction partners governing the multifunctionality of potyviral proteins. Intrinsically disordered domains/regions of these proteins play an important role in their interactions with other proteins. Deciphering the function of PVBV-encoded proteins and their interactions with cognitive partners will help in understanding the putative mechanisms by which the potyviral proteins are regulated at different stages of the viral life-cycle. This review also discusses PVBV virus-like particles (VLPs) and their potential applications in nanotechnology. Further, virus-like nanoparticle-cell interactions and intracellular fate of PVBV VLPs are also discussed

Biological Significance of Marine Actinobacteria of East Coast of Andhra Pradesh, India

An attempt was made to identify actinobacterial strains present in the marine soil of East Coast regions viz., Chirala, Bapatla, and Peddaganjam, Andhra Pradesh; Kanyakumari, Tamil Nadu and Goa, Goa along with the study of their antimicrobial potential. Eight out of 73 actinobacterial strains isolated from these regions showed strong antimicrobial activity against Gram positive bacteria, Gram negative bacteria, and Candida albicans. Molecular identification (16S rRNA analysis) of the eight strains revealed that they belong to Dietzia sp., Kocuria sp., Nocardiopsis sp., and Streptomyces spp. ISP (International Streptomyces project) -1, ISP-2 and starch casein media supported high antimicrobial potential after 5-6 days of growth. Production of antimicrobials by the strains varied significantly with different carbon and nitrogen sources. Gas chromatography mass spectrometry (GCMS) analysis of volatile compounds produced by the strains illustrated an array of antimicrobial compounds such as 1, 2-benzene dicarboxylic acid, 2-piperidinone, pyrrolo1,2-a] pyrazine-1,4-dion, phenyl ethyl alcohol, 3-phenyl propionic acid etc. Ours is the first report on the study and detection of above mentioned antimicrobial metabolites from Dietzia sp. (A3), Kocuria sp. (A5), and Nocardiopsis sp. (A7). By sequence based analysis for secondary metabolites, non-ribosomal peptide synthetase (NRPS) gene cluster was noticed in six strains (A2, A3, A4, A6, A7, and A8) and none of them had polyketide synthase (PKS) system. The present study intimates the biological potentiality of the actinobacterial strains isolated from East Coast of Andhra Pradesh, India which reveals further scope to investigate new bioactive compounds from them by employing both natural product chemistry and modern biotechnological aspects