Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily.
The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.
Daubner
S Colette
SC
Departments of Biochemistry and Biophysics and Chemistry, Texas A&M University, College Station, TX 77843-2128, USA.
Gadda
Giovanni
G
Valley
Michael P
MP
Fitzpatrick
Paul F
PF
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000145:classification / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D049308:Dioxygenases; D004926:Escherichia coli; D005670:Fusarium / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000145:classification / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase