2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Purification and some properties of the enzyme.
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 non-aromatic compound. 2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 mumol.min-1.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 less than or equal to 25 microM) and O2 (Km less than or equal to 5 microM), but less specific for the reduced pyridine nucleotides NADH (Km = 42 microM) or NADPH [Km = 500 microM; 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.
Buder
R
R
Abteilung Angewandte Mikrobiologie, Universität Ulm, FRG.
Fuchs
G
G
D002384:Catalysis; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000032:analysis; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D011549:Pseudomonas / Q000201:enzymology*; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D005420:Flavoproteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C062023:2-aminobenzoyl-CoA monooxygenase-reductase