Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.
FEMS Microbiol Lett
; 365(16)2018 08 01.
Article
en En
| MEDLINE
| ID: mdl-29931366
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 (kcat of 5.91 ± 0.18 s-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. 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
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Campylobacter jejuni
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Secuencia de Aminoácidos
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Clonación Molecular
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Periplasma
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Nitrato-Reductasa
Idioma:
En
Revista:
FEMS Microbiol Lett
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos