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Type: Journal article
Title: Improving the affinity and activity of CYP101D2 for hydrophobic substrates
Author: Bell, S.
Yang, W.
Dale, A.
Zhou, W.
Wong, L.
Citation: Applied Microbiology and Biotechnology, 2013; 97(9):3979-3990
Publisher: Springer-Verlag
Issue Date: 2013
ISSN: 0175-7598
Statement of
Stephen G. Bell, Wen Yang, Alison Dale, Weihong Zhou, Luet-Lok Wong
Abstract: CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).
Keywords: Cytochrome P450 monooxygenase
Novosphingobium aromaticivorans
C–H bond activation
Protein engineering
Rights: Copyright Springer-Verlag 2012
DOI: 10.1007/s00253-012-4278-7
Appears in Collections:Aurora harvest
Chemistry publications

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