title: Variations in the stability of NCR ene reductase by rational enzyme loop modulation.
abstract: The engineering of protein stability is of major importance for the application of enzymes in a wide range of industrial applications. Here we study the determinants of the thermo- and solvent stability of the Zymomonas mobilis ene reductase NCR using a rational protein engineering approach based on analyses of structural and sequence data. We designed and created two loop mutants with the aim to increase their overall stability. They all retained catalytic activity but exhibited altered thermostability relative to the wild-type enzyme. The modulation of one specific loop segment near the active site of NCR showed an increased tolerance to organic solvents along with an enhanced thermostability.
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Reich
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Kress
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Bettina M
authors.initials: BM
authors.lastname: Nestl
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Electronic address: bernhard.hauer@itb.uni-stuttgart.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003512:Cyclohexanones / Q000737:chemistry; D004795:Enzyme Stability; D038181:FMN Reductase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012997:Solvents / Q000737:chemistry; D017355:Zymomonas / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D003512:Cyclohexanones; D012997:Solvents; C476259:ketoisophorone; D038181:FMN Reductase
keywords: Enzyme engineering; Old Yellow Enzyme; Rational loop design; Solvent stability; ThermoFAD; Thermostability