title: Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme.
abstract: A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to benzoyl-CoA. The further downstream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl-CoA is oxidized to 2,3-dihydro-2,3-dihydroxybenzoyl-CoA (benzoyl-CoA dihydrodiol) by benzoyl-CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxC(mal)) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3-dihydro-2,3-dihydroxybenzoyl-CoA + H(2)O --> 3,4-dehydroadipyl-CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring-(13)C(6)]benzoyl-CoA, benzoyl-CoA oxygenase/reductase, BoxC(mal) protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde; the missing one-carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde and [2,3,4,5,6-(13)C(5)]4,5-dehydroadipyl-CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed benzoyl-CoA-dihydrodiol lyase. It contains domains characteristic for enoyl-CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring-cleavage reaction does not require molecular oxygen.
authors.affiliation: Institut für Biologie II, Mikrobiologie, Fakultät für Biologie, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Gescher
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eisenreich
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wörth
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Georg
authors.initials: G
authors.lastname: Fuchs
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000332:Aerobiosis; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012663:Semicarbazides / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D004952:Esters; D012663:Semicarbazides; D013438:Sulfhydryl Compounds; C037817:benzoyl-coenzyme A; D008190:Lyases