Universität zu Köln

Mintmier, Breeanna, McGarry, Jennifer M., Sparacino-Watkins, Courtney E., Sallmen, Joseph, Fischer-Schrader, Katrin ORCID: 0000-0003-4532-8918, Magalon, Axel, McCormick, Joseph R., Stolz, John F., Schwarz, Guenter ORCID: 0000-0002-2118-9338, Bain, Daniel J. and Basu, Partha (2018). Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni. FEMS Microbiol. Lett., 365 (16). OXFORD: OXFORD UNIV PRESS. ISSN 1574-6968

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Abstract

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (k(cat) of 5.91 +/- 0.18 s(-1) and a K-M (nitrate) of 3.40 +/- 0.44 mu M) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Mintmier, Breeanna UNSPECIFIED UNSPECIFIED UNSPECIFIED McGarry, Jennifer M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Sparacino-Watkins, Courtney E. UNSPECIFIED UNSPECIFIED UNSPECIFIED Sallmen, Joseph UNSPECIFIED UNSPECIFIED UNSPECIFIED Fischer-Schrader, Katrin UNSPECIFIED orcid.org/0000-0003-4532-8918 UNSPECIFIED Magalon, Axel UNSPECIFIED UNSPECIFIED UNSPECIFIED McCormick, Joseph R. UNSPECIFIED UNSPECIFIED UNSPECIFIED Stolz, John F. UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwarz, Guenter UNSPECIFIED orcid.org/0000-0002-2118-9338 UNSPECIFIED Bain, Daniel J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Basu, Partha UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-177695
DOI:	10.1093/femsle/fny151
Journal or Publication Title:	FEMS Microbiol. Lett.
Volume:	365
Number:	16
Date:	2018
Publisher:	OXFORD UNIV PRESS
Place of Publication:	OXFORD
ISSN:	1574-6968
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language DIMETHYL-SULFOXIDE REDUCTASE; SITE-DIRECTED MUTAGENESIS; ESCHERICHIA-COLI; MOLYBDENUM COORDINATION; SUBSTRATE-BINDING; ELECTRON-TRANSFER; AMINO-ACIDS; PURIFICATION; SEQUENCE; IDENTIFICATION Multiple languages Microbiology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/17769