title: Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast.
abstract: Previously, N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway of L-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-related N-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library of MPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increase L-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higher L-arginine synthetic activity and also improved fermentation ability.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nasuno
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Saeka
authors.initials: S
authors.lastname: Hirase
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Norifune
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Watanabe
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan hiro@bs.naist.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism*; D001383:Azetidinecarboxylic Acid / Q000494:pharmacology; D057075:Enzyme Assays; D004795:Enzyme Stability; D005285:Fermentation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013696:Temperature
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D001383:Azetidinecarboxylic Acid; D001120:Arginine; D000123:Acetyltransferases; C492447:Mpr1 protein, S cerevisiae
keywords: N-acetyltransferase Mpr1; l-arginine biosynthesis; structure-based molecular design; thermostability; yeast