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

Item Type:Article
Title:Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16
Creators Name:Siddiqui, R.A. and Warnecke-Eberz, U. and Hengsberger, A. and Schneider, B. and Kostka, S. and Friedrich, B.
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 of A. 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.
Keywords:Alcaligenes, Amino Acid Sequence, Base Sequence, Molecular Cloning, Bacterial DNA, Polyacrylamide Gel Electrophoresis, Bacterial Genes, Molecular Sequence Data, Mutation, Nitrate Reductase, Nitrate Reductases, Phenotype, Amino Acid Sequence Homology, Structure-Activity Relationship
Source:Journal of Bacteriology
ISSN:0021-9193
Publisher:American Society for Microbiology (U.S.A.)
Volume:175
Number:18
Page Range:5867-5876
Date:1 September 1993
Official Publication:http://jb.asm.org/cgi/content/abstract/175/18/5867
PubMed:View item in PubMed

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