title: Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering.
abstract: A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening). Thus ∼ 20,000 protein variants could be screened in summary of which five were found to transform DPM with a specific activity of up to 75% of the wild-type activity on d-camphor and a coupling rate of 7-18%. One variant converting DPM to 4-hydroxydiphenylmethane (4HDPM) was subjected to site-directed mutagenesis and saturation mutagenesis, which revealed the particular importance of positions F87, Y96 and L244 for substrate selectivity and the possibility for further improvements of this variant. Moreover, a reduction in size of the amino acid at position 396 decreased specific activity dramatically but increased coupling and switched the main product formation from 4HDPM towards diphenylmethanol.
authors.affiliation: Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Hoffmann
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bönsch
authors.forename: Thomas
authors.initials: T
authors.lastname: Greiner-Stöffele
authors.forename: Meike
authors.initials: M
authors.lastname: Ballschmiter
mesh_terms: D001559:Benzhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D055808:Drug Discovery; D006900:Hydroxylation; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001559:Benzhydryl Compounds; D019475:Camphor 5-Monooxygenase; C010129:diphenylmethane