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

Abstract

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H2O2 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 Angstroms 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 Angstroms resolution. The c axis for the trigonal space group of oxidized NAO is 485 Angstroms, and there are six subunits (11/2 holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E{sup ox}). The active-site structures of E{sup 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 inmore » 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 Angstroms. 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.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914312
Report Number(s):
BNL-78880-2007-JA
Journal ID: ISSN 0006-2960; TRN: US200809%%169
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 45
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CRYSTAL STRUCTURE; ELECTRON DENSITY; OXIDASES; REACTION KINETICS; SPACE GROUPS; national synchrotron light source

Citation Formats

Nagpal,A., Valley, M., Fitzpatrick, P., and Orville, A. Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism of a Covalent Complex of the Flavoenzyme Trapped During Turnover. United States: N. p., 2006. Web. doi:10.1021/bi051966w.
Nagpal,A., Valley, M., Fitzpatrick, P., & Orville, A. Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism of a Covalent Complex of the Flavoenzyme Trapped During Turnover. United States. doi:10.1021/bi051966w.
Nagpal,A., Valley, M., Fitzpatrick, P., and Orville, A. Sun . "Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism of a Covalent Complex of the Flavoenzyme Trapped During Turnover". United States. doi:10.1021/bi051966w.
@article{osti_914312,
title = {Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism of a Covalent Complex of the Flavoenzyme Trapped During Turnover},
author = {Nagpal,A. and Valley, M. and Fitzpatrick, P. and Orville, A.},
abstractNote = {Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H2O2 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 Angstroms 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 Angstroms resolution. The c axis for the trigonal space group of oxidized NAO is 485 Angstroms, and there are six subunits (11/2 holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E{sup ox}). The active-site structures of E{sup 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 Angstroms. 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.},
doi = {10.1021/bi051966w},
journal = {Biochemistry},
number = ,
volume = 45,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}