Publications

• Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii.

  Drevland R.M., Waheed A., Graham D.E.

  The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. Th ... >> More

  The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence. << Less

  J. Bacteriol. 189:4391-4400(2007) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Absolute configuration of alpha isopropylmalate and the mechanism of its conversion to beta isopropylmalate in the biosynthesis of leucine.

  Cole F.E., Kalyanpur M.G., Stevens C.M.

  Biochemistry 12:3346-3350(1973) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes.

  Jang S., Imlay J.A.

  An Escherichia coli strain that cannot scavenge hydrogen peroxide has been used to identify the cell processes that are most sensitive to this oxidant. Low micromolar concentrations of H2O2 completely blocked the biosynthesis of leucine. The defect was tracked to the inactivation of isopropylmalat ... >> More

  An Escherichia coli strain that cannot scavenge hydrogen peroxide has been used to identify the cell processes that are most sensitive to this oxidant. Low micromolar concentrations of H2O2 completely blocked the biosynthesis of leucine. The defect was tracked to the inactivation of isopropylmalate isomerase. This enzyme belongs to a family of [4Fe-4S] dehydratases that are notoriously sensitive to univalent oxidation, and experiments confirmed that other members were also inactivated. In vitro and in vivo analyses showed that H2O2 directly oxidized their solvent-exposed clusters in a Fenton-like reaction. The oxidized cluster then degraded to a catalytically inactive [3Fe-4S] form. Experiments indicated that H2O2 accepted two consecutive electrons during the oxidation event. As a consequence, hydroxyl radicals were not released; the polypeptide was undamaged; and the enzyme was competent for reactivation by repair processes. Strikingly, in scavenger-deficient mutants, the H2O2 that was generated as an adventitious by-product of metabolism (<1 microm) was sufficient to damage these [4Fe-4S] enzymes. This result demonstrates that aerobic organisms must synthesize H2O2 scavengers to avoid poisoning their own pathways. The extreme vulnerability of these enzymes may explain why many organisms, including mammals, deploy H2O2 to suppress microbial growth. << Less

  J. Biol. Chem. 282:929-937(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• THE BIOSYNTHESIS OF LEUCINE. II. THE ENZYMIC ISOMERIZATION OF BETA-CARBOXY-BETA-HYDROXYISOCAPROATE AND ALPHA-HYDROXY-BETA-CARBOXYISOCAPROATE.

  GROSS S.R., BURNS R.O., UMBARGER H.E.

  Biochemistry 2:1046-1052(1963) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.