title: Site Saturation Mutagenesis Applications onCandida methylicaFormate Dehydrogenase.
abstract: In NADH regeneration,Candida methylicaformate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependentcmFDH from NAD+to NADP+and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability ofcmFDH, the conserved residue at position 1 in the catalytic domain ofcmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating "smarter libraries" for improving the properties ofcmFDH.
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey; Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey.
authors.forename: Gülşah P
authors.initials: GP
authors.lastname: Özgün
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey; Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey; Department of Molecular Biology and Genetics, Faculty of Science and Letters, Yıldız Technical University, Istanbul, Turkey.
authors.forename: Emel B
authors.initials: EB
authors.lastname: Ordu
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey; Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey.
authors.forename: H Esra
authors.initials: HE
authors.lastname: Tütüncü
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey; Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey.
authors.forename: Emrah
authors.initials: E
authors.lastname: Yelboğa
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol, Avon BS8 1TD, UK.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey; Istanbul Technical University Molecular Biology-Biotechnology & Genetics Research Center, 34469 Istanbul, Turkey.
authors.forename: Nevin
authors.identifier: 0000-0002-2300-9484
authors.initials: N
authors.lastname: Gül Karagüler