Kinetic and mechanistic studies on the reactions of 2-aminobenzoyl-CoA monooxygenase/reductase.

The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase with its natural substrates 2-aminobenzoyl-CoA, NADH and O2 has been investigated using the stopped-flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2-aminobenzoyl-CoA and NADH, a turnover number of approximately 40 min-1 was found at pH 7.4 and 4 degrees C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a approximately 1:1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2-amino benzoyl-CoA to reduced enzyme is, in contrast, a simple second-order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2-aminobenzoyl-CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH2 state. This rate is not affected by the presence of 2-aminobenzoyl-CoA. In contrast, 2-aminobenzoyl-CoA enhances approximately 100-fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs = 2.0 s-1) is partially rate-limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate-complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin-4a-hydroxide. Thus, insertion of oxygen into 2-aminobenzoyl-CoA is essentially complete at this point and has a kobs > or = 80 s-1. The subsequent phase is accompanied by formation of the main product, 2-amino-5-oxocyclohex-1-enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non-aromatic CoA intermediate; it has a rate approximately 1.3 s-1, which is thus the second rate-limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kobs approximately 0.05 s-1). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-1-enecarboxyl-CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2-amino-5-hydroxybenzoyl-CoA as secondary product. The reduced enzyme formed in this process is reoxidized by O2 to form H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Aminobenzoyl-CoA monooxygenase/reductase. Evidence for two distinct loci catalyzing substrate monooxygenation and hydrogenation.

2-Aminobenzoyl-CoA monooxygenase/reductase catalyzes both monooxygenation and hydrogenation of anthraniloyl-CoA. Its reactivity with 11 substrate analogs has been investigated. Only 2-aminobenzoyl-CoA (anthraniloyl-CoA) in its normal and deuterated (5-2H) form is a full substrate, and only traces of 2-hydroxybenzoyl-CoA (salicyloyl-CoA) are probably monooxygenated but not hydrogenated. The purified enzyme is a homodimer and has been resolved preparatively into three major species by anion-exchange chromatography on Mono Q. All three species have the same specific activity when reconstituted to full content of FAD, they differ, however, substantially in their mode of binding FAD. The oxidized or fully reduced enzyme forms bind tightly 0.5 mol/mol of the substrate 2-aminobenzoyl-CoA (Kd = 1-2 microM). The enzyme can be depleted of approximately 50% of its FAD, which corresponds to essentially complete removal from one of the two binding sites, reflecting a large difference in the affinity for FAD. From this it is deduced that the two sites are not equivalent. Removal of FAD from one binding site leads to loss of the hydrogenation capacity of the enzyme, while monooxygenation catalysis is retained. The FAD cofactors of the two binding sites differ drastically in their reactivities towards NADH, oxygen and N-ethylmaleimide. Exchange of reducing equivalents between the two FAD cofactors at the respective binding sites is very slow and irrelevant compared to the rates of catalysis. It is concluded that the enzyme, which has been proposed to consist of two identical polypeptide chains [Altenschmidt, U., Bokranz, M. & Fuchs, G. (1992) Eur. J. Biochem. 207, 715-722], contains two active centers which differ substantially in their catalytic activity. One center belongs to the class of monooxygenases, the other one to the (de)hydrogenases. This must result from substantially different interaction of the same flavin cofactors with protein functional groups and is, to our knowledge, unprecedented in flavoprotein enzymology.

Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis.

The reaction of two D-amino acid oxidases from the yeasts Rhodotorula gracilis and Trigonopsis variabilis with the substrates alanine and valine in their 2-1H and 2-2H forms was studied employing the stopped-flow spectrophotometric technique. The turnover numbers at infinite substrate and oxygen concentrations were: 20,700/4,250 and 1,730/360 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,150/440 and 2,500/520 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The rates of anaerobic enzyme flavin reduction were 20,100/4,000 and 1,820/350 ([2-1H]/[2-2H]alanine and valine, respectively) for the Rhodotorula and 3,470/350 and 2,460/480 ([2-1H]/[2-2H]alanine and valine, respectively) for the Trigonopsis enzymes. The isotope effects on enzyme reduction were 5.0 and 5.2 for Rhodotorula and 9.9 and 5.1 for Trigonopsis D-amino acid oxidases with alanine and valine, respectively. This suggests that the intrinsic isotope effect on rupture of the substrate alpha-C-H bond can be as high as 10. The rate-determining step corresponds to the enzyme reductive half-reaction in contrast to the mammalian kidney enzyme where it is the product release from oxidized enzyme (Massey, V., and Gibson, Q.H. (1964) Fed. Proc. 23, 18-29). Upon anaerobic reaction with substrate, the yeast enzymes do not form the transient long wavelength absorbing species which are characteristic of the mammalian protein. This is due only in part to rapid dissociation of iminoacid product and is ascribed to intrinsic differences between the charge-transfer complexes of reduced enzyme flavin and product of the yeast as compared to the mammalian enzyme. With the Trigonopsis enzyme the flavin radical anion appears to be strongly stabilized and can be produced quantitatively.

2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Identification of the reaction products.

In a previous report we have described some properties of a novel flavoenzyme from a denitrifying Pseudomonas species which catalyzes the oxygen- and NAD(P)H-dependent conversion of 2-aminobenzoyl-CoA [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur. J. Biochem. 185, 637-634]. In this paper, we report on the identification of the three products formed from 2-aminobenzoyl-CoA in this reaction. The spectroscopic data and the chemical properties of these compounds and those of their degradation products are compatible with the structures of 2-amino-5-hydroxybenzoyl-CoA, 2-amino-5-hydroxycyclohex-1-enecarboxyl-CoA and of 2-amino-5-oxocyclohex-1-enecarboxyl-CoA. The latter is the main product and was found to be rather unstable since it hydrolyzes and decarboxylates readily at pH less than 5. Ammonia is released from the decarboxylation product in the neutral pH range to yield 1,4-cyclohexanedione. Conditions were optimized such that the CoA thioester of 2-amino-5-hydroxybenzoate is the product obtained at greater than 98% yield. 2-amino-5-hydroxycyclohex-1-enecarboxyl-CoA is the product which is formed when the mixture of the reaction products is treated with sodium borohydride before separation.

2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Studies on the stoichiometry and the course of the reaction.

The reaction catalyzed by 2-aminobenzoyl-coenzyme-A monooxygenase/reductase from a denitrifying Pseudomonas sp. has been investigated. 2-Aminobenzoyl-CoA and 2-amino[carboxy-14C]benzoyl-CoA were synthesized enzymatically using 2-aminobenzoyl-CoA synthetase from the same organism. The product was purified by chromatography and characterized by ultraviolet/visible and 1H-NMR spectroscopy. The conversion of 2-aminobenzoyl-CoA catalyzed by the monooxygenase/reductase requires NADH and oxygen, and yields at least two different products depending on the relative concentration of NADH. At [NADH] less than Km (40 microM), i.e. [NADH]/[2-aminobenzoyl-CoA] approximately 0.02-0.05, the main product is probably a hydroxylated derivative of 2-aminobenzoyl-CoA, which is characterized by an absorbance maximum around 375 nm. When [NADH]/[2-aminobenzoyl-CoA] approximately 2-5, the predominant product is a non-aromatic coenzyme A thioester (lambda max approximately 320 nm). The stoichiometry in this case is 2.1-2.4 mol NADH oxidized (mol oxygen consumed)-1 (mol 2-aminobenzoyl-CoA metabolized)-1. The product is extremely unstable in the acidic pH range and undergoes decarboxylation in a few minutes at pH less than 5. Some degree of stabilisation is obtained upon reduction with sodium borohydride, probably resulting in a further reduced non-aromatic coenzyme-A thioester.