Involvement of a Flavosemiquinone in the Enzymatic Oxidation of Nitroalkanes Catalyzed by 2-Nitropropane Dioxygenase

Abstract

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1-and propyl-2-nitronate, for which a nonenzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k cat /K m determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[ 2 H 4 ]nitroethane and 1,1,2-[ 2 H 3 ]ethyl nitronate at pH 8 yielded normal and inverse effects on the k cat /K m value, respectively, and were negligible on the k cat value. The k cat /K m and k cat pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.2-Nitropropane dioxygenase (EC 1.13.11.32) from Neurospora crassa is a flavin-dependent enzyme, which catalyzes the oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrite. The enzyme was first characterized by Little (1) in 1951 as an oxidase, but was later classified as a dioxygenase based on the observation that the oxygen atom of the organic product formed upon oxidation of propyl-2-nitronate originates from molecular oxygen and not from water (2). To date, 2-nitropropane dioxygenase has been isolated from N. crassa (1) and Hansenula mrakii (3). Whereas the two enzymes have similar molecular weights of about 40,000, they differ in their prosthetic group content in that...

Crystal Structure of 2-Nitropropane Dioxygenase Complexed with FMN and Substrate

“…The recombinant enzyme has a native molecular mass of ϳ37 kDa (calculated monomer mass including the C-terminal tag ϭ 35,884 Da), as estimated by our dynamic light scatter-ing analysis. This result indicates that P. aeruginosa 2-nitropropane dioxygenase is monomeric in solution, unlike the homodimeric 2-nitropropane dioxygenase from N. crassa (16).…”

“…It has been shown that 2-nitropropane dioxygenase operates through an oxidase-style catalytic mechanism, in which substrate oxidation occurs prior to and independently from reaction with oxygen, via the formation of an enzyme-bound anionic flavin semiquinone (16). This represents the first account in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for the oxidation of organic molecules catalyzed by a flavindependent enzyme (16).…”

“…This represents the first account in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for the oxidation of organic molecules catalyzed by a flavindependent enzyme (16).…”

“…FMN and FAD are found in the N. crassa and W. mrakii (H. mrakii) enzymes, respectively (14,15). The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16).…”

“…The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16). A steady-state kinetic analysis showed that the preferred substrates for the enzyme are anionic nitronates as compared with neutral nitroalkanes and that the enzyme has broad substrate specificity that is independent of substrate size (16).…”