Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability.
The budding yeast Saccharomyces cerevisiae Sigma1278b has the MPR1 gene, which confers resistance to the proline analogue azetidine-2-carboxylate (AZC). This gene encodes an N-acetyltransferase Mpr1 that detoxifies AZC, and the homologous genes have been found in many yeasts. Recently, we found that Mpr1 protects yeast cells by reducing the intracellular reactive oxygen species (ROS) levels under oxidative stresses, such as heat-shock, freezing, or ethanol treatment. Unlike the known antioxidant enzymes, Mpr1 is thought to acetylate toxic metabolite(s) involved in ROS generation via oxidative events. To improve the enzymatic functions of Mpr1, we applied PCR random mutagenesis to MPR1. The mutagenized plasmid library was introduced into the S. cerevisiae S288C strain lacking MPR1, and we successfully isolated two Mpr1 variants with higher AZC resistance (K63R and F65L/L117V). Interestingly, overexpression of the K63R variant was found to increase cell viability or decrease intracellular ROS levels after exposure to H(2)O(2) or ethanol compared with the wild-type Mpr1. In vitro studies with the recombinant enzymes showed that the catalytic efficiency of the K63R variant for AZC and acetyl-CoA was higher than that of the wild-type Mpr1 and that the F65L mutation greatly enhanced the thermal stability. The mutational analysis and molecular modeling suggest that an alpha-helix containing Lys63 and Phe65 has important roles in the function of Mpr1. In addition, the wild-type and K63R variant Mpr1 reduced intracellular ROS levels under ethanol stress conditions on haploid sake yeast cells. These results suggest that engineering Mpr1 might be useful in breeding oxidative stress-tolerant yeast strains.
Iinoya
Kaoru
K
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Kotani
Tetsuya
T
Sasano
Yu
Y
Takagi
Hiroshi
H
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001384:Azetidines / Q000378:metabolism*; D004795:Enzyme Stability; D018628:Gene Dosage; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020125:Mutation, Missense; D016877:Oxidants / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D001384:Azetidines; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; C089382:azetidyl-2-carboxylic acid; D006861:Hydrogen Peroxide; D000123:Acetyltransferases; C492447:Mpr1 protein, S cerevisiae