Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha.
Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.
Hettmann
Thomas
T
Institute for Molecular Biotechnology, Beutenbergstr. 11, 07745 Jena, Germany.
Siddiqui
Roman A
RA
Frey
Christa
C
Santos-Silva
Teresa
T
Romão
Maria João
MJ
Diekmann
Stephan
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000596:Amino Acids; D008982:Molybdenum; D005560:Formate Dehydrogenases; D009565:Nitrate Reductases; D050901:Nitrate Reductase