Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

Abstract

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His(6)-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K ( m ) values for nitrite and NADH were 0.5 mM and 20 microM, respectively, at the pH and temperature optima (pH 9 and 30 degrees C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.

The Tetrapyrrole Biosynthetic Pathway and Its Regulation in Rhodobacter capsulatus

“…VitB 12 and its derivatives are the most complex tetrapyrrole synthesized in nature, and are involved in various cellular functions such as DNA repair and methionine synthesis (Martens et al 2002). The synthesis of siroheme in a R. capsulatus strain has been reported as well (Olmo-Mira et al 2006). …”

“…A 17 kb region contains genes encoding an assimilatory nitrate reduction system, which consists in: (i) putative regulatory genes nsrR and nasTS ; (ii) an ABC-type nitrate transporter coded by nasFED ; (iii) genes coding for the apo-nitrate and -nitrite reductases, nasA and nasB ; (iv) a gene encoding a siroheme synthase, cysG , responsible of synthesizing the cofactor of the nitrite reductase (Pino et al 2006). Although the siroheme synthase was not biochemically studied, the nitrite reductase NasB was heterologously expressed in E. coli with purified recombinant protein exhibiting a spectral signature of siroheme (Olmo-Mira et al 2006). CysG (EC 2.1.1.107) was characterized in Salmonella enterica and in E. coli where it was shown to be a bimodular homodimer that catalyses the four reactions that converts uro’gen III into siroheme (Stroupe et al 2003).…”

Anaerobic phototrophic nitrite oxidation by Thiocapsa sp. strain KS1 and Rhodopseudomonas sp. strain LQ17

“…Nonetheless, nitrogen compounds are used in assimilatory or dissimilatory metabolism. Although ammonia is usually their preferred nitrogen source, some phototrophic strains assimilate nitrate or nitrite if ammonia is absent (Malofeeva et al, 1974;Klemme, 1979;Pino et al, 2006;Olmo-Mira et al, 2006). Furthermore, fixation of molecular nitrogen is common among most anoxygenic phototrophs (Gogotov & Glinskii, 1973;Malofeeva & Laush, 1976;Madigan et al 1984).…”

UNRAVELING THE REGULATION OF NITROGEN ASSIMILATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE): DIURNAL VARIATIONS IN TRANSCRIPT LEVELS FOR FIVE GENES INVOLVED IN NITROGEN ASSIMILATION1

“…Bacterial and fungal nir B holoenzymes are homodimers with FAD‐ and NAD(P)H‐dependent functions at the N‐terminal end of the protein and NO 2 − ‐reducing functions near the C‐terminus (Colandene and Garrett 1996). The enzymes contain a well‐conserved iron–sulfur half domain (2Fe–2S), a nitrite/sulfite reductase ferredoxin‐like half domain, and binding sites for two prosthetic groups: iron–sulfur (4Fe–4S) and siroheme (Colandene and Garrett 1996, Olmo‐Mira et al. 2006).…”