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Allen Orville
's research interests explained in more detail:
Nitroalkane Oxidase (NAO & NAO-ES*)
Nitrite elimination reactions from nitroaliphatics, which are produced by plants to inhibit TCA cycle
enzymes in pathogens, provide a survival advantage to some organisms. For example, nitroalkane oxidase (NAO),
an FAD-dependent enzyme in fungus is induced by nitroalkanes and enables the microbe to obtain all its
nitrogen from these types of compounds. Oxidized NAO readily crystallizes in a trigonal space group, diffracts
to beyond 1.6 Å, but with a unit cell c edge of ~485 Å. To determine the structure we used crystals of a
Se-Met enriched NAO, but trapped as a stable reaction intermediate (NAO-ES*). This intermediate is trapped at
low temperature during NAO turnover of nitroethane as an N5-(2-nitrobutyl)-1,5-dihydro-FAD adduct. Although the
orthorhombic unit cell of NAO-ES* is smaller than oxidized NAO, it nevertheless, required analysis of 52 Se
sites with MAD phasing methods. Moreover, this 2.2 Å structure is the first one for a flavoenzyme trapped
turnover of a true substrate. The structure of NAO-ES* was also used to solve the structure of oxidized NAO
in the large unit cell, which has been refined to 2.07 Å resolution. The overall protein fold of NAO is
similar to several acyl-CoA dehydrogenases (ACAD), but the substrate access channel differs between NAO and
the ACAD homologues. Moreover, the structure for the trapped intermediate in NAO supports the carbanion-based
reaction mechanism proposed for NAO and differentiates it from the hydride transfer mechanism proposed for the
acyl-CoA dehydrogenase family members.
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Supported by: American Chemical Society, Petroleum Research Fund (40310-G4), A.M.O. (PI) and
American Heart Association Grant in Aid (0555286B), A.M.O. (PI).
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Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms.
Acta Crystallography, D60:1456-1460 (2004).
PubMed
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Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
Crystal structures of nitroalkane oxidase: High resolution data collection strategy for long cell edged crystals.
NSLS Science Highlights,
2004 NSLS Activity Report (2005).
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Fitzpatrick P.F., Orville A.M., Nagpal A. and Valley M.P.
Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase.
Arch Biochem Biophys., 433:157-165 (2005).
PubMed
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Orville A.M., Nagpal A., Manning L., Blehert D.S., Valley M.P., Chambliss G.H., Fox B.G., and Fitzpatrick P.F.
Structural Perspective on Nitrite Elimination of Organic Nitrochemicals by Flavoenzymes.
in Flavins and Flavoproteins 2005, (T. Nishino, R. Miura, M. Tanokura, K. Fukui, edts)
ARchiTect Inc., 827-840 (2005).
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Fitzpatrick P.F., Valley M.P., Gadda G., Nagpal A. and Orville A.M.
The Mechanism of Nitroalkane Oxidase.
in Flavins and Flavoproteins 2005, (T. Nishino, R. Miura, M. Tanokura, K. Fukui, edts)
ARchiTect Inc., 59-69 (2005).
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Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism from a Covalent
Complex of the Flavoenzyme Trapped during Turnover.
Biochemistry, 45:1138-1150 (2006).
PubMed
PDB files:
2C12,
2C0U
Last Modified: April 10, 2009
Please forward all questions about this site to:
Denise Monteleone
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