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Multiple sequence alignment of homoserine-acetyltransferases, serine-acetyltransferases and homoserine-succinyltransferases show they all belong to MetX family, having evolved from a common ancestor by conserving the catalytic site and substrate binding residues. The discrimination in the substrate selection arises due to the presence of substrate-specific residues lining the substrate-binding pocket. Mutation of Ala59 and Gly62 to Gly and Pro respectively in homoserine-acetyltransferase from M. tuberculosis resulted in a serine-acetyltransferase like enzyme as it acetylated both l-homoserine and l-serine. Homoserine-acetyltransferase from M. tuberculosis when mutated at positon 322 where Leu was converted to Arg, resulted in succinylation over acetylation of l-homoserine. Our studies establish the importance of the substrate binding residues in determining the type of activity possessed by MetX family, despite all of them having the same catalytic triad Ser-Asp-His. Hence key residues at the substrate binding pocket dictate whether the given enzyme shows predominant transferase or hydrolase activity.

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The metA (Rv3341) gene from Mycobacterium tuberculosis H37Rv strain encodes a homoserine-acetyltransferase (HAT) enzyme, also called MetA. This enzyme plays a key role in the biosynthetic pathway of methionine and is a potential target for the development of antimicrobial drugs. Purified MetA showed 40 kDa molecular mass on SDS-PAGE. Manual docking was performed with substrates acetyl-CoA, l-homoserine, and p-nitrophenylacetate using crystal structure coordinates of MetA (PDB ID 6PUX) from M. tuberculosis. Multiple sequence alignment indicated that catalytic triad residues Ser157, Asp320, His350 were conserved across species in acetyltransferases, esterases, and hydrolases. As a conserved pentapeptide, GXSMG belongs to α/ß hydrolase superfamily and it shares similarity with esterases and hydrolases from different sources. Hydrolase activity of MetA was tested using (PNPA), N-acetylglycine, N-acetylmethionine and Phe-Gly as substrate. LC-MS confirmed that MetA possessed HAT activity, but no homoserine-succinyltransferase (HST) and serine-acetyltransferase (SAT) activities. Replacing acetyl-CoA with PNPA as acetyl group donor showed a drastic reduction in transferase activity, arising due to the interaction of R227 of the enzyme with PNPA. This could prevent the binding of the second substrate in the right orientation and results in the preferential transfer of the acetyl group to water, thus exhibiting hydrolase rather than transferase activity. In this paper, we report that MetA has both transferase and hydrolase activity depending on the correct orientation of the second substrate and the availability of the amino acids involved in enzyme-substrate interaction.

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The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120CHD +ATP.Ca2+ ), 5D0E (Ma1120CHD +GTP.Ca2+ ), 5D0H (Ma1120CHD (KDA→EGY)+ATP.Ca2+ ), 5D0G (Ma1120CHD (KDA→EGY)+GTP.Ca2+ ). ENZYMES: Adenylyl cyclase (EC number: 4.6.1.1).

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An adenylyl cyclase from Mycobacterium avium, Ma1120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Ma1120 in a monomeric form and its truncated construct as a dimer. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional α-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions.

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Mutational, crystallographic and phylogenetic analysis of nucleotidyl cyclases have been used to understand how these enzymes discriminate between substrates. Ma1120, a class III adenylyl cyclase (AC) from Mycobacterium avium, was used as a model to study the amino acid residues that determine substrate preference, by systematically replacing ATP specifying residues with those known to specify GTP. This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH. Replacement of key residues lysine (101) and aspartate (157) with residues conserved across GCs by site directed mutagenesis, led to a marked improvement in GC activity and a decrease in AC activity. This could be correlated to the presence and strength of the hydrogen bond between the second substrate binding residue (157) and the base of the nucleotide triphosphate. This is substantiated by the fact that the pH optimum is highly dependent on the amino acid residues present at positions 101 and 157.

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Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn(2+) ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn(2+) ions in SACOL0085.

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Intravenous injection of gelonin and deglycosylated gelonin led to rapid clearance from the blood. Both molecules distributed similarly in liver and kidney suggesting that they followed the same pathway. Deglycosylation reduced the uptake by a third in liver, but did not affect uptake by kidney. Studies with Triton WR1339 showed a classical lysosomal pathway for both molecules. The deglycosylated molecule was degraded to a greater extent than native gelonin as seen by the presence of acid soluble radioactivity. Cell separation showed that while endothelial cells mainly took up native gelonin, Kupffer cells took up the deglycosylated molecule.

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Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.

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