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Rational engineering of the fungal P450 monooxygenase CYP5136A3 to improve its oxidizing activity toward polycyclic aromatic hydrocarbons.

Protein engineering, design & selection : PEDS (2013-08-02)
Khajamohiddin Syed, Aleksey Porollo, David Miller, Jagjit S Yadav
ABSTRACT

A promising polycyclic aromatic hydrocarbon-oxidizing P450 CYP5136A3 from Phanerochaete chrysosporium was rationally engineered to enhance its catalytic activity. The residues W129 and L324 found to be critical in substrate recognition were transformed by single (L324F) and double (W129L/L324G, W129L/L324F, W129A/L324G, W129F/L324G and W129F/L324F) mutations, and the engineered enzyme forms were expressed in Pichia pastoris. L324F and W129F/L324F mutations enhanced the oxidation activity toward pyrene and phenanthrene. L324F also altered the regio-selectivity favoring C position 4 over 9 for hydroxylation of phenanthrene. This is the first instance of engineering a eukaryotic P450 for enhanced oxidation of these fused-ring hydrocarbons.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phenanthrene, 98%
Supelco
Phenanthrene, analytical standard, for environmental analysis
Sigma-Aldrich
Phenanthrene, sublimed grade, ≥99.5%
Supelco
Phenanthrene, certified reference material, TraceCERT®
Pyrene, BCR®, certified reference material
Sigma-Aldrich
Pyrene, puriss. p.a., for fluorescence, ≥99.0% (GC)
Sigma-Aldrich
Pyrene, 98%
Supelco
Phenanthrene solution, certified reference material, 5000 μg/mL in methanol
Supelco
Pyrene, analytical standard
Sigma-Aldrich
Pyrene, sublimed grade, 99%
Supelco
Pyrene, certified reference material, TraceCERT®