Publications

• Biochemical and structural elucidation of the L-carnitine degradation pathway of the human pathogen <i>Acinetobacter baumannii</i>.

  Piskol F., Lukat P., Kaufhold L., Heger A., Blankenfeldt W., Jahn D., Moser J.

  <i>Acinetobacter baumannii</i> is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. S ... >> More

  <i>Acinetobacter baumannii</i> is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the <i>in vitro</i> and <i>in vivo</i> reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)⁺-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys²⁹⁰ and Glu²⁵⁶, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg¹⁶⁶, Arg²⁸⁴ and Ser⁴⁴⁷ while dual cofactor specificity for NAD⁺ and NADP⁺ is mediated by Asn¹⁸⁴. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD⁺-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a β-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire <i>A. baumannni</i> pathway was heterologously reconstituted, allowing <i>E. coli</i> to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO₂ and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health. << Less

  Front Microbiol 15:1446595-1446595(2024) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• 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.

• Oxidation D-malic and beta-alkylmalic acids wild-type and mutant strains of Salmonella typhimurium and by Aerobacter aerogenes.

  Stern J.R., O'Brien R.W.

  A mutant strain of Salmonella typhimurium (SL 1634 dml-51) capable of growth on d-malate as sole carbon source was shown to produce d-malic enzyme. This enzyme was absent in the parent wild-type strain which was unable to grow on d-malate. Growth of the mutant on d-malate also resulted in a greatl ... >> More

  A mutant strain of Salmonella typhimurium (SL 1634 dml-51) capable of growth on d-malate as sole carbon source was shown to produce d-malic enzyme. This enzyme was absent in the parent wild-type strain which was unable to grow on d-malate. Growth of the mutant on d-malate also resulted in a greatly increased level of beta-isopropylmalic enzyme compared with its level in the wild-type strain grown on citrate or l-malate. The d-malic and beta-isopropylmalic enzymes, both of which catalyze a nicotinamide adenine dinucleotide- and Mg(++)-dependent oxidative decarboxylation of their respective substrates, were shown to be distinct enzymes by selective inhibition with erythro-dl-beta-hydroxyaspartate and by other methods. Cell extracts of the mutant strain also oxidized dl-beta-methyl-, dl-beta-ethyl-, dl-beta-propyl- and dl-betabeta-dimethylmalates, in order of decreasing activity. dl-beta-Methyl-malate was shown to be oxidized by both the d-malic and the beta-isopropylmalic enzymes, whereas the oxidation of the other beta-alkylmalates appeared to be effected exclusively by the beta-isopropylmalic enzyme. beta-Isopropylmalic enzyme activity was induced by d-malate but not by l-malate, showing that it behaved as a d-malictype enzyme. Growth of Aerobacter aerogenes on d-malate, which caused induction of d malic enzyme, resulted in only a small increase in the activity of beta-isopropylmalic enzyme. << Less

  J Bacteriol 98:147-151(1969) [PubMed] [EuropePMC]

• Characterization of the multiple catalytic activities of tartrate dehydrogenase.

  Tipton P.A., Peisach J.

  Tartrate dehydrogenase (TDH) has been purified to apparent homogeneity from Pseudomonas putida and has been demonstrated to catalyze three different NAD(+)-dependent reactions. TDH catalyzes the oxidation of (+)-tartrate to form oxaloglycolate and the oxidative decarboxylation of D-malate to form ... >> More

  Tartrate dehydrogenase (TDH) has been purified to apparent homogeneity from Pseudomonas putida and has been demonstrated to catalyze three different NAD(+)-dependent reactions. TDH catalyzes the oxidation of (+)-tartrate to form oxaloglycolate and the oxidative decarboxylation of D-malate to form pyruvate and CO2. D-Glycerate and CO2 are formed from meso-tartrate in a reaction that is formally a decarboxylation with no net oxidation or reduction. The steady-state kinetics of the first two reactions have been investigated and found to follow primarily ordered mechanisms. The pH dependence of V and V/K was determined and indicates that catalysis requires that a base on the enzyme with a pK of 6.7 be unprotonated. TDH activity requires a divalent and a monovalent cation. Kinetic data suggest that the cations function in substrate binding and facilitation of the decarboxylation of beta-ketoacid intermediates. << Less

  Biochemistry 29:1749-1756(1990) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.