Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha

Biochemical and Biophysical Research Communications
Thomas HettmannStephan Diekmann

Abstract

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.

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Sep 27, 2003·Biochemical and Biophysical Research Communications·Thomas HettmannStephan Diekmann
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Citations

Jul 20, 2007·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Matthias Hofmann
Mar 23, 2011·Journal of Molecular Biology·Catarina CoelhoMaria João Romão
Feb 5, 2013·Biochimica Et Biophysica Acta·Stéphane GrimaldiAxel Magalon
Jan 29, 2014·Chemical Reviews·Russ HillePartha Basu
Nov 2, 2020·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Breeanna MintmierPartha Basu
Jan 18, 2006·Journal of Inorganic Biochemistry·P J GonzálezJ J G Moura

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