Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability

Abstract

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.

Structure-based molecular design for thermostabilization ofN-acetyltransferase Mpr1 involved in a novel pathway of l-arginine synthesis in yeast

“…The results showed that the remaining activity of each variant decreased with the incubation time. The half-life of the activity of Phe65Leu-Mpr1 (32.2 ± 9.0 min) was longer than that of the WT-Mpr1 (11.4 ± 4.8 min), as previously reported (12). Interestingly, Asn203Lys-Mpr1 and Asn203Arg-Mpr1 similarly exhibited 2.4-fold (27.0 ± 1.0 min) and 2.2-fold (23.8 ± 4.8 min) longer activity half-lives than WT-Mpr1.…”

“…Previously, we isolated an Mpr1 variant with higher thermostability, Phe65Leu-Mpr1, by the screening from PCR random mutagenesis library of MPR1 (12). Recently, we determined the three-dimensional structure of Mpr1 by crystallographic analysis (13), and showed that the folding pattern of Mpr1 is similar to those of proteins in the Gcn5-related N-acetyltransferase (GNAT) superfamily, to which Mpr1 also belongs.…”

Structural and functional analysis of the yeast N -acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

“…Interestingly, the Lys63Arg variant confers higher oxidative stress tolerance than WT-Mpr1 on yeast cells. The Phe65Leu mutation enhanced the thermal stability of Mpr1 (29). In the structure of Mpr1, Phe65 is buried in the hydrophobic region (Fig.…”

“…Previously, we isolated two Mpr1 variants with higher AZC resistance (Lys63Arg, Phe65Leu) (29). Interestingly, the Lys63Arg variant confers higher oxidative stress tolerance than WT-Mpr1 on yeast cells.…”

“…Because Mpr1 confers AZC resistance on various organisms, selectable marker systems using the MPR1 gene and AZC have been constructed for yeast and plant transformation (33)(34)(35). We recently showed that industrial yeast strains expressing the Mpr1 variant are more tolerant to oxidative stresses, such as ethanol, freezing, and desiccation, than those expressing WT-Mpr1 (29,36,37). Engineered Mpr1 might be useful in breeding oxidative stress-tolerant yeast strains with improved fermentation ability.…”