Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16

Abstract

Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability ofA. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.Nitrate is a major nitrogen source for many bacteria. In the general assimilatory pathway, nitrate is converted via nitrite to ammonia, which is then assimilated into nitrogen metabolism. This metabolic route functions aerobically and anaerobically and involves assimilatory nitrate reductases which are repressed by ammonia. Nitrate can also serve as an electron acceptor for anaerobic respiration in the absence of oxygen. In this case nitrate is reduced by respiratory nitrate reductases to nitrite (reviewed in references 39 and 46). In a variety of bacteria, includingAlcaligenes eutrophus H16 (24), the end product of nitrate respiration is dinitrogen. This denitrification pathway involves, in addition to the respiratory nitrate reductase, further respiratory reductases for nitrite, nitric oxide, and nitrous oxide (reviewed in reference 46).A. eutrophus H16 is a gram-negative obligate respiratory bacterium which is able to grow heterotrophically on a broad range of substrates. Alternatively, it can grow autotrophically with hydrogen as an energy source, and in the absence of oxygen it can use nitrate or nitrite for anaerobic respiration (8). These alternative facultative metabolic pathways of A. eutrophus H16 are genetically linked to the megaplasmid pHG1 (13,31). Physiological and genetic ...

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures

“…For example, Nap plays a role in anaerobic denitrification in Pseudomonas G-179 and Rhodobacter sphaeroides f. sp. denitrificans, in anaerobic nitrate respiration under nitrate-limited conditions in E. coli, and redox-poising of the photosynthetic electrontransport chain or survival during light-dark transitions in Rhodobacter capsulatus (Jones et al, 1990;Siddiqui et al, 1993;Castillo et al, 1996;Zumft, 1997;Liu et al, 1999;Moreno-Vivian et al, 1999;Philippot & Hojberg, 1999;Potter et al, 1999Potter et al, , 2001Richardson et al, 2001;Brondijk et al, 2002;Ellington et al, 2003). Previously, the importance of transcriptional regulation for the regulation of Nap activity in P. pantotrophus has been established in batch culture (Ellington et al, 2002).…”

Purification and Characterization of the Selenate Reductase from Thauera selenatis

“…Selenate reductase is dissimilar to the periplasmic nitrate reductases that have been purified from Rhodobacter sphaeroides, Alcaligenes eutrophus, and Thiosphaera pantotropha (3,(25)(26)(27). These enzymes consist of two subunits containing molybdopterin, possibly one [Fe-S] center and cytochrome c as prosthetic groups.…”

“…Selenate is an abundant oxyanion in some habitats, particularly in agricultural drainage waters from seleniferous soils (1)(2)(3)(4). High concentrations of selenium oxyanions can have detrimental effects on wildlife such as bird species in the Kesterson Reservoir (5,6).…”

Characterization of the paramagnetic iron‐containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase

“…The enzyme from Thiosphaera pantotropha has been most extensively characterized [1,2] but the enzyme has also been purified from Rhodobacter sphaeroides f.sp. denitrificans [3], Rhodobacter capsulatui [4-61 and Alcaligenes eutrophus [7]. In T pantotropha, the enzyme comprises a 90 kDa molybopterin-binding catalytic subunit (NapA) and a 16 kDa cytochrome-c,,, (NapB).…”