Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N5,N10-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT

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

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of l-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ∼5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ-phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity

Functional insights into the role of C-terminal disordered domain of Sesbania mosaic virus RNA-dependent RNA polymerase and the coat protein in viral replication in vivo

The C-terminal disordered domain of sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) interacts with the viral protein P10. The functional significance of this interaction in viral replication was examined by a comparative analysis of genomic and sub-genomic RNA levels (obtained by quantitative real time PCR) in the total RNA extracted from Cyamopsis plants agro-infiltrated with wild-type or mutant forms of SeMV infectious cDNA (icDNA). The sgRNA copy numbers were found to be significantly higher than those of gRNA in the wild-type icDNA transfected plants. Transfection of a mutant icDNA expressing an RdRp lacking the Cterminal disordered domain led to a drastic reduction in the copy numbers of both forms of viral RNA. This could be due to the loss of interaction between the disordered domain of RdRp and P10 and possibly other viral/host proteins that might be required for the assembly of viral replicase. The C-terminal disordered domain also harbours the motif E which is essential for the catalytic function of RdRp. Mutation of the conserved tyrosine within this motif in the full length icDNA resulted in complete inhibition of progeny RNA synthesis in the transfected plants confirming the importance of motif E in the polymerase function in vivo. The role of coat protein (CP) in viral infection was also investigated by agro-infiltration of a CP start codon mutant icDNA which suggested that CP is essential for the encapsidation of viral progeny RNAs at later stages of infection

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement

Interaction of the intrinsically disordered C-terminal domain of the sesbania mosaic virus RNA-dependent RNA polymerase with the viral protein P10 in vitro: modulation of the oligomeric state and polymerase activity

The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 mu M using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro

"Natively Unfolded" VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus ($\triangle$N70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the $\triangle$N70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the $\triangle$N70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta

Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is one among the deadliest pathogens in the world. Due to long treatment regimen, HIV co-infection, persistence of bacilli in latent form and development of XDR and TDR strains of Mtb, tuberculosis has posed serious concerns for managing the disease, and calls for discovery of new drugs and drug targets. Using a computational pipeline involving analysis of the structural models of the Mtb proteome and an analysis of the ATPome, followed by a series of filters to identify druggable proteins, solubility and length of the protein, several candidate proteins were shortlisted. From this, Rv3405c, a tetR family of DNA binding protein involved in antibiotic resistance, was identified as one of the good drug targets. Rv3405c binds to the upstream non coding region of Rv3406 and causes repression of Rv3406 activity there by affecting the downstream processes involved in antibiotic resistance was further characterized. The Rv3405c gene was cloned; the gene product was over-expressed in E. coli and purified by Ni NTA chromatography. DNA binding studies by EMSA showed that the recombinant Rv3405c protein binds to the DNA sequence corresponding to the promoter region of Rv3406 and upon addition of tetracycline, the DNA binding activity was lost. P-galactosidase reporter assay in E. coli using both wild type and a DNA binding defective mutant protein indeed proved that Rv3405c acts as a repressor. (C) 2018 Elsevier Inc. All rights reserved

Production of biosurfactant “Biosur-Pm” by Pseudomonas maltophila CSV89: characterization and role in hydrocarbon uptake

Pseudomonas maltophilia CSV89, a soil bacterium, produces an extracellular biosurfactant, ''Biosur-Pm''. The partially purified product is nondialyzable and chemically composed of 50% protein and 12-15% sugar, which indicates the complex nature of Biosur-Pm. It reduces the surface tension of water from 73 to 53 x 10(-3) N m(-1) and has a critical micellar concentration of 80 mg/l. Compared to aliphatic hydrocarbons, Biosur-Pm shows good activity against aromatic hydrocarbons. The emulsion formed is stable and does not require any metal ions for emulsification. The kinetics of Biosur-Pm production suggest that its synthesis isa growth-associated and pH-dependent process. At pH 7.0, cells produced more Biosur-Pm with less cell surface hydrophobicity. At pH 8.0, however, the cells produced less Biosur-Pm with more cell surface hydrophobicity and showed a twofold higher affinity for aromatic hydrocarbons compared to the cells grown at pH 7.0. The Biosur-Pm showed a pH-dependent release, stimulated growth of the producer strain on mineral salts medium with 1-naphthoic acid when added externally, and facilitated the conversion of salicylate to catechol. All these results suggest that Biosur-Pm is probably a cell-wall component and helps in hydrocarbon assimilation/uptake