Functional characterization of constituent enzyme fractions of mycobacillin synthetase.
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ABSTRACT:
SUBMITTER: Ghosh SK
PROVIDER: S-EPMC1152684 | biostudies-other | 1985 Sep
REPOSITORIES: biostudies-other
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Project description:The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.
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Project description:Mycobacillin synthetase lacks aspartic acid racemase, alanine racemase and glutamic acid racemase activities. The enzyme also does not respond to ATP-[32P]Pi exchange, nor does it catalyse the antibiotic synthesis in presence of amino acids of configuration opposite to that present in the molecule. Preincubation with optical isomers of opposite configuration inhibited the ATP-[32P]Pi exchange reaction to the extent of 60-90%. None of the three fractions of mycobacillin synthetase contained a pantothenic acid arm. Two molecules of ATP are required to synthesize one peptide bond of mycobacillin. Intermediate peptides of mycobacillin are not covalently linked to the three-fraction mycobacillin synthetase.
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Project description:The extracellular release of mycobacillin from Bacillus subtilis first occurred in the medium at the onset of stationary phase and continued at a high rate even after 6 days. Mycobacillin synthetase activity appeared earlier than late-exponential phase in the cytosol of producer cells and was not sedimentable even at 105 000 g. The activity then quickly reached the maximum late in the stationary phase. With further increase in the age of the culture, the activity gradually disappeared from the cytosol, to reappear concomitantly in the membrane in an insoluble particulate form, even in absence of protein synthesis. The membrane-bound synthetase activity was sedimentable at 10 000 g and was fairly active even after 5 days.
| S-EPMC1144638 | biostudies-other
Project description:The mycobacillin-synthesizing enzyme system was highly purified by fractionation at 30-55% (NH4)2SO4 saturation. The enzyme concentrate on Sephadex G-200 gel chromatography was resolved into three distinct fragments. Each of the fragments on further purification by DEAE-cellulose ion-exchange chromatography behaved as a single-component system, as clearly indicated by the sharpness of the peaks in the elution diagram. None of the fragments alone nor any two of them in all possible combinations possessed mycobacillin-synthesizing activity, which was restored only when the three fragments were used together in the test system.
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Project description:Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNA(Pro). Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNA(Pro), whereas Cys-tRNA(Pro) is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNA(Pro) deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2'-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein's conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis.
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Project description:Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a ?2 type and a ?2?2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both ?2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1- strain, while Escherichia coli GlyRS (a ?2?2-type enzyme) and A. thaliana organellar GlyRS (a (??)2-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail ?? fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a ?2-type eukaryotic GlyRS may be functionally substituted with a ?2?2-type bacterial cognate enzyme despite their remote evolutionary relationships.
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Project description:Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.
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Project description:Mammalian lipoxygenases play a role in normal cell development and differentiation but they have also been implicated in the pathogenesis of cardiovascular, hyperproliferative and neurodegenerative diseases. As lipid peroxidizing enzymes they are involved in the regulation of cellular redox homeostasis since they produce lipid hydroperoxides, which serve as an efficient source for free radicals. There are various epidemiological correlation studies relating naturally occurring variations in the six human lipoxygenase genes (SNPs or rare mutations) to the frequency for various diseases in these individuals, but for most of the described variations no functional data are available. Employing a combined bioinformatical and enzymological strategy, which included structural modeling and experimental site-directed mutagenesis, we systematically explored the structural and functional consequences of non-synonymous genetic variations in four different human lipoxygenase genes (ALOX5, ALOX12, ALOX15, and ALOX15B) that have been identified in the human 1000 genome project. Due to a lack of a functional expression system we resigned to analyze the functionality of genetic variations in the hALOX12B and hALOXE3 gene. We found that most of the frequent non-synonymous coding SNPs are located at the enzyme surface and hardly alter the enzyme functionality. In contrast, genetic variations which affect functional important amino acid residues or lead to truncated enzyme variations (nonsense mutations) are usually rare with a global allele frequency<0.1%. This data suggest that there appears to be an evolutionary pressure on the coding regions of the lipoxygenase genes preventing the accumulation of loss-of-function variations in the human population.
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