2‐Aminobenzoyl‐CoA monooxygenase/reductase, a novel type of flavoenzyme

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Abstract
A novel aerobic mechanism of 2-aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2-Aminobenzoic acid is activated in a coenzyme-A-ligase reaction to 2-aminobenzoyl-CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2-aminobenzoyl-CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown nonaromatic compound. 2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 .mu.mol.cntdot.min-1.cntdot.mg-1 protein using ammonium sulfate precipitation, DEAE-cellulose anion-exchange, hydroxylapatite and Mono Q FPLC anion-exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an .alpha.2 dimer; however, it may exist in three dimeric forms, .alpha..alpha., .alpha..alpha.'' and .alpha.''.alpha.'', where .alpha. may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2-aminobenzoyl-CoA (Km .ltoreq. 25 .mu.M) and O2 (Km .ltoreq. 5 .mu.M), but less specific for the reduced pyridine nucleotides NADH (Km = 42 .mu.M) or NADPH [Km = 500 .mu.M; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min-1. The enzyme also reduced N-ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.