New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii.
A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways. In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl-CoA, which is subsequently attacked by an oxygenase, followed by a non-oxygenolytic fission of the ring. We provide evidence for this hypothesis and show that benzoyl-CoA conversion in the bacterium Azoarcus evansii requires NADPH, O(2) and two protein components, BoxA and BoxB. BoxA is a homodimeric 46 kDa iron-sulphur-flavoprotein, which acts as reductase. In the absence of BoxB, BoxA catalyses the benzoyl-CoA stimulated artificial transfer of electrons from NADPH to O(2) via free FADH(2) to produce H(2)O(2). Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron-protein that acts as benzoyl-CoA oxygenase. The product of benzoyl-CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA. This suggests that BoxBA act as a benzoyl-CoA dioxygenase/reductase. Unexpectedly, benzoyl-CoA transformation by BoxBA was greatly stimulated when another enoyl-CoA hydratase/isomerase-like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl-CoA. The benzoyl-CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.
Zaar
Annette
A
Institut für Biologie II, Mikrobiologie, Universität Freiburg, Schänzlestr.1, D-79104 Freiburg, Germany.
Gescher
Johannes
J
Eisenreich
Wolfgang
W
Bacher
Adelbert
A
Fuchs
Georg
G
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000332:Aerobiosis; D000595:Amino Acid Sequence; D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000378:metabolism; D016384:Consensus Sequence; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D001565:Benzoates; D009249:NADP; C037817:benzoyl-coenzyme A; D010105:Oxygenases; D003066:Coenzyme A Ligases; D010100:Oxygen