Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni

Breeanna Mintmier (1) , Jennifer Mcgarry (1) , Courtney Sparacino-Watkins (2) , Joseph Sallmen (3) , Katrin Fischer-Schrader (4) , Axel Magalon (5) , Joseph Mccormick (3) , John Stolz (3) , Günter Schwarz (4) , Daniel Bain (6) , Partha Basu (1)

1 IUPUI - Indiana University - Purdue University Indianapolis
2 PITT - University of Pittsburgh
3 Duquesne University [Pittsburgh]
4 University of Cologne
5 LCB - Laboratoire de chimie bactérienne
6 Department of Geology and Environmental Science, University of Pittsburgh, PA 15260, USA.

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, Michaelis-Menten kinetics (kcat of 5.91 ± 0.18s - 1 and a KM (nitrate) of 3.40± 0.44 μM) 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. jejunion nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating residue, cysteine has been exchanged for serine. The resulting variant NapA is four fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represents the first isolation and characterization of an Epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Keywords: Campylobacter; Heterologous Expression; NapA; Nitrate Reductase; Pathogenicity; Molybdenum

Domains: Life Sciences [q-bio]

Breeanna Mintmier, Jennifer Mcgarry, Courtney Sparacino-Watkins, Joseph Sallmen, Katrin Fischer-Schrader, et al.. Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni. FEMS Microbiology Letters, 2018, 365 (16), ⟨10.1093/femsle/fny151⟩. ⟨hal-01919478⟩