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CRYSTAL STRUCTURES OF NITROALKANE OXIDASE: INSIGHTS INTO THE STRUCTURAL BASIS FOR SUBSTRATE SPECIFICITY AND THE CATALYTIC MECHANISM

by Nagpal, Akanksha

Abstract (Summary)
Nitrochemicals are widely used as explosives, biocides and drugs. In addition, 3-nitro-tyrosine and other nitrated protein residues are important markers for many cardiovascular, neurodegenerative, and malignant conditions. Because of the wide presence of the nitrocompounds as toxins, potential nitrogen/carbon sources, and metabolic intermediates, different organisms have evolved to produce enzymes that can biodegrade nitrocompounds. The structural studies of the enzymes, which catalyze the removal of nitro group from nitrochemicals, are of considerable interest for both applied and fundamental reasons. The insights into the reaction mechanism of these enzymes can be used for designing efficient biocatalysts for bioremediation and for developing antibiotics for disease resistant microbes. Nitroalkane oxidase (NAO) produced by Fusarium oxysporum is a novel flavoenzyme that catalyzes the oxidation of the neutral nitroalkanes to the corresponding aldehydes or ketones with production of H2O2 and nitrite. Sequence analysis of NAO shows that it belongs to acyl-CoA dehydrogenase (ACAD) superfamily but the enzyme does not catalyze acyl-CoA substrates and follows a different reaction mechanism. The structural studies of different reaction states of NAO were undertaken to define the basis for the unique carbanion mechanism and substrate specificity. A 2.2 Å resolution crystal structure of a substrate trapped reaction intermediate of NAO (ES*) was solved by MAD phasing with 52 SeMet sites. The structure of ES* was used to solve the crystal structure of the oxidized NAO (P3221; c-axis 485 Å) to 2.07 Å resolution. Four out of the six active sites in the oxidized NAO crystal structure contain spermine (EI), a weak competitive inhibitor; the other two subunits are empty (Eox).The crystal structures of Eox, EI and ES* reveal a hydrophobic N5 substrate access channel with Asp402 positioned on the re-face of the flavin adenine dinucleotide (FAD). The structural overlays of NAO and ACADs exhibit differences in substrate access channel and in hydrogen bonding patterns between the respective active site base, substrate molecules and the FAD in the active site. These differences are likely to distinguish NAO from its homologs and are proposed to form the basis of the unique reaction mechanism of NAO.
Bibliographical Information:

Advisor:Dr. Allen M. Orville; Dr. Loren D. Williams; Dr. Donald F. Doyle; Dr. Dale E. Edmondson; Dr. Giovanni Gadda

School:Georgia Institute of Technology

School Location:USA - Georgia

Source Type:Master's Thesis

Keywords:chemistry and biochemistry

ISBN:

Date of Publication:07/19/2005

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