Purification and properties of nitroalkane oxidase from Fusarium oxysporum

Abstract

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium, was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(N02)CH2CH3 + 02 + H20 + OHCCH2CH3 + HNO2 + H202. In addition to 1-nitropropane, 3-nitro-2-pentanoL 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavine adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavine 5'phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and Nethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavine adenine dinucleotide, 1.33 uM.

Amazonian soil metagenomes indicate different physiological strategies of microbial communities in response to land use change

“…In addition to differences in methane cycling, our results highlighted differences in pathways of nitrogen cycling. Higher gene content to nitrify nitroalkanes in the forest suggests that nitroalkanes may serve as sole sources of nitrogen, carbon, and energy in its microbial communities (52,53). Our results also suggest that nitrifying archaeal phylum Thaumarchaeota and several species of it are more frequently distributed in forest soils, which was also shown in our previous study using functional marker genes (24).…”

Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism from a Covalent Complex of the Flavoenzyme Trapped during Turnover

“…NAO from F. oxysporum has been very well-characterized in recent years (reviewed in ref 20). The enzyme is produced in large quantities when cultures are grown on nitroethane (9), and reasonable physiological roles for the enzyme include (a) protecting the fungus from nitroalkane toxins produced by plants (6), (b) detoxifying antibiotics produced by the competing soil microbes such as Pseudomonas fluorescens (21), and/or (c) scavenging nitrogen from a variety of nitrochemicals. The molecular weight of the 439-residue monomeric subunit is 47 955, but analytical ultra-centrifugation suggests that the enzyme exists in solution as a homotetramer/homodimer mixture (22).…”

Nitroalkane Oxidation by Streptomycetes

“…Nitroalkane-oxidizing activity has been reported in many microorganisms (6,9,12,17), but so far only 2-nitropropane dioxygenase from H. mrakii (10,11,13), a /3-nitropropionate-oxidizing enzyme from Penicilliuni atrotenetum (6) and Aspergillus flatus (23), and a nitroalkane oxidase from Fusarium oxysporium (9) which yields nitrite, acetone, and hydrogen peroxide from 2-nitropropane and oxygen have been studied in more or less detail. We have considered the possibility that these three enzymes or a number of nonspecific enzymes, such as i)amino acid oxidase (30), glucose oxidase (29), glutathione S-transferase (7), and possibly xanthine dehydrogenase, could be responsible for the observed nitroalkane oxidations in the crude cell-free extracts of the Streptomnyces strains investigated.…”

“…On the basis of these microbiological studies, it appears that nitrosation might proceed quite readily either as an enzyme-catalyzed process or possibly as an or-ganic matter-catalyzed process (5,22), provided that nitrous acid can be generated within the respective cells. Two main pathways that can lead to the formation of nitrous acid have been reported in microorganisms: reduction of inorganic nitrates (26) and degradation of organic nitro compounds (6,9,12,17,23,33). The first pathway is probably not a general route in Streptomyces strains for nitrous acid formation since many of them cannot grow on nitrate as the sole nitrogen source.…”