Related Experiment Video
Updated: Jun 4, 2026

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products
Published on: October 4, 2019
Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering
Gregor Hoffmann1, Kathrin Bönsch, Thomas Greiner-Stöffele
1Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.
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.
Related Concept Videos
Pharmacogenetics of Phase I Enzymes: Cytochrome P450 Isozymes
Reduction of Alkenes: Asymmetric Catalytic Hydrogenation
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
C4 Pathway and CAM
C4 Pathway
The C4 pathway is used by plants such as...
Introduction to Mechanisms of Enzyme Catalysis
Factors Affecting Drug Biotransformation: Physicochemical and Chemical Properties of Drugs
The drug's acidity or basicity is essential in determining the metabolic...
Enzyme Inhibition

