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.

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases.

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R.

Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme.

A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg). The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit.

Metal-mediated reactions modeled after nature.

The Collaborative Research Center (CRC) 436 'Metal-Mediated Reactions Modeled after Nature' was founded for the express purpose of analyzing the catalytic principles of metallo-enzymes in order to construct efficient catalysts on a chemical basis. The structure of the active center and neighboring chemical environment in enzymes serves as a focal point for developing reactivity models for the chemical redesign of catalysts. Instead of simply copying enzyme construction, we strive to achieve new chemical intuition based on the results of long-lasting natural evolution.