title: Effects of mutation at position 285 of Ralstonia pickettii T1 poly[(R)-3-hydroxybutyrate] depolymerase on its activities.
abstract: Asn at position 285 (N285) in the catalytic domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 most likely participates in the cleavage of ester bonds as revealed by our previous evolutionary engineering study using the error-prone polymerase chain reaction (PCR) method. To exhaustively examine the effects of mutations at that position, we conducted site-directed saturation mutagenesis at that position and the resultant mutant enzymes (N285X) were evaluated in p-nitrophenyl ester (pNPCn) hydrolysis and PHB degradation. Kinetic studies demonstrated that the PHB-degrading activities of N285X were reciprocally related to their pNPCn-hydrolyzing activities, with the exception of N285A and N285G, and that His residue could functionally substitute for Asn285 on PHB degradation.
authors.affiliation: Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan.
authors.forename: Liu-Tzea
authors.initials: LT
authors.lastname: Tan
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Mizuo
authors.initials: M
authors.lastname: Maeda
mesh_terms: D019943:Amino Acid Substitution; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011091:Polyesters / Q000378:metabolism*; D049920:Ralstonia pickettii / Q000201:enzymology* / Q000235:genetics; D000094122:Polyhydroxybutyrates
chemical_list: D002265:Carboxylic Ester Hydrolases; D006885:Hydroxybutyrates; D050505:Mutant Proteins; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase