Biochemistry. 2006 Jan 31;45(4):1138-50.

Crystal structures of nitroalkane oxidase: insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover.

Nagpal A, Valley MP, Fitzpatrick PF, Orville AM.

Source

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.

Abstract

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.

PMID:: 16430210; [PubMed - indexed for MEDLINE]
PMCID:: PMC1855086

Free PMC Article

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Figure 6

Comparison of substrate-binding channels in NAO and the MCAD–C8S complex. (A) Divergent stereoview of the solvent-accessible surfaces around the FAD in subunit A of the NAO E^ox active site. The FAD is shown as CPK atoms with carbon, nitrogen, oxygen, and phosphorus atoms colored green, blue, red, and magenta, respectively. The active-site base, Asp402, as well as Phe401 and Phe273 are shown as sticks with carbon atoms colored yellow. The surfaces are colored according to the atoms that form the border and with gray surfaces from subunit A and purple surfaces from subunit B. The N5 channel extends from the bottom right to the right of α-helix G and terminates under the Phe401–Asp402 residues. The N1 channel extends from the bottom left, under the α-helix K, and terminates near Phe273 from α-helix G. (B) N5 substrate-access channel in NAO is occupied by a spermine molecule in subunit B of oxidized NAO (EI). For a comparison, several residues from the superimposed structure of the MCAD–C8S complex (PDB code 1udy) are shown in cyan. The NAO residues are labeled and shown in green, and the FAD residues are labeled and shown in orange. (C) Corresponding solvent-accessible surface areas in the MCAD–C8S complex. The MCAD residues are labeled and colored as in B. For a comparison, several residues from the superimposed structure of NAO–EI are shown in green. The MCAD–C8S cavities are interrupted by Thr255 and blocked from the exterior by His65. (D) Residues that form hydrogen bonds to the C8S molecule in the MCAD–C8S complex (magenta) and the corresponding residues from NAO (green, residue names with asterisks). (E) Some of the residues that constrict the N1 channel in NAO include Phe273, Leu408, and Arg415 and the corresponding residues in the MCAD–C8S complex.

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

Biochemistry. 2006 January 31;45(4):1138-1150.