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Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of
neutral nitroalkanes to the corresponding aldehydes or ketones with the
production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the
acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA
substrates. We present the 2.2 A resolution crystal structure of NAO trapped
during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The
homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites
in an orthorhombic space group. The electron density for the
N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The
structure of ES was used to solve the crystal structure of oxidized NAO at 2.07
A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A,
and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four
of the active sites contain spermine (EI), a weak competitive inhibitor, and two
do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and
ES* reveal a hydrophobic channel that extends from the exterior of the protein
and terminates at Asp402 and the N5 position on the re face of the FAD. Thus,
Asp402 is in the correct position to serve as the active-site base, where it is
proposed to abstract the alpha proton from neutral nitroalkane substrates. The
structures for NAO and various members of the ACAD family overlay with
root-mean-square deviations between 1.7 and 3.1 A. The homologous region
typically spans more than 325 residues and includes Glu376, which is the
active-site base in the prototypical member of the ACAD family. However, NAO and
the ACADs exhibit differences in hydrogen-bonding patterns between the
respective active-site base, substrate molecules, and FAD. These likely
differentiate NAO from the homologues and, consequently, are proposed to result
in the unique reaction mechanism of NAO.
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