Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues

Marshall, Samantha Jane, Krause, Doreen, Blencowe, Dayle K. and White, Graham Francis 2004. Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues. Journal of Bacteriology 186 (6) , pp. 1802-1810. 10.1128/JB.186.6.1802-1810.2004

Full text not available from this repository.

Abstract

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 kcat as that of wild-type enzyme but with altered Km values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of KmGTN to KmNAD(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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
ISSN:	0021-9193
Last Modified:	31 Jan 2020 06:31
URI:	https://orca.cardiff.ac.uk/id/eprint/1079

Citation Data

Cited 14 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item