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Journal of Bacteriology, March 2004, p. 1802-1810, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1802-1810.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues

Samantha J. Marshall, Doreen Krause, Dayle K. Blencowe, and Graham F. White^*

School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom

Received 2 October 2003/ Accepted 2 December 2003

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10^-7 M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k_cat as that of wild-type enzyme but with altered K_m values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K_m^GTN to K_m^NAD(P)H, with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.

Present address: Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom.

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