title: Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172.
abstract: Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoyl-CoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-electron reduction step, possibly yielding cyclohex-1,5-diene-1-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium Thauera aromatica strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP, both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yield under anaerobic conditions in the presence of 0.25 mM dithionite. It had a native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maximum at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35,000 M-1 cm-1. Reduction of the enzyme by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The enzyme contained 10.8 +/- 1.5 mol Fe and 10.5 +/- 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or flavin-like compound; the estimated content was 0.3 mol/mol enzyme. The enzyme reaction required MgATP and a strong reductant such as Ti(III). The reaction catalyzed is: benzoyl-CoA + 2 Ti(III) + n ATP-->non-aromatic acyl-CoA + 2 Ti(IV) + n ADP + n Pi. The estimated number n of ATP molecules hydrolyzed/two electrons transferred in benzoyl-CoA reduction is 2-4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg(2+)-ATP, other nucleoside triphosphates being inactive (< 1%). Mg2+ could be substituted to some extent by Mn2+, Fe2+ and less efficiently by Co2+. Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 mumol min-1 mg-1 corresponding to a catalytic number of 1.6 s-1. The apparent Km values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were 15 microM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, bipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine (Km 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation energy for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP.
authors.affiliation: Angewandte Mikrobiologie, Universität Ulm, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Boll
authors.forename: G
authors.initials: G
authors.lastname: Fuchs
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000693:Anaerobiosis; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005030:Ethylenes / Q000494:pharmacology; D019143:Evolution, Molecular; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology; D006868:Hydrolysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010269:Paraquat / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D005030:Ethylenes; D006898:Hydroxylamines; D019811:Hydroxylamine; C037817:benzoyl-coenzyme A; D000255:Adenosine Triphosphate; C036216:ethylene; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C109030:benzoyl-coenzyme A reductase (dearomatizing); D010269:Paraquat