Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering
| Autor: | Thomas Greiner-Stöffele, Gregor Hoffmann, Meike Ballschmiter, Kathrin Bönsch |
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| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Camphor 5-Monooxygenase Diphenylmethanol Stereochemistry Bioengineering Diphenylmethane Hydroxylation Protein Engineering Biochemistry Substrate Specificity chemistry.chemical_compound Catalytic Domain Drug Discovery Benzhydryl Compounds Saturated mutagenesis Molecular Biology biology Chemistry Pseudomonas putida Substrate (chemistry) Active site Stereoisomerism Protein engineering Monooxygenase Mutagenesis Mutation biology.protein Specific activity Biotechnology |
| Zdroj: | Protein engineering, designselection : PEDS. 24(5) |
| ISSN: | 1741-0134 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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