Title: Benign synthesis of 2-ethylhexanoic acid by cytochrome P450cam: enzymatic, crystallographic, and theoretical studies.

Authors: French, K J; Strickler, M D; Rock, D A; Rock, D A; Bennett, G A; Wahlstrom, J L; Goldstein, B M; Jones, J P

Published In Biochemistry, (2001 Aug 14)

Abstract: This study examines the ability of P450cam to catalyze the formation of 2-ethylhexanoic acid from 2-ethylhexanol relative to its activity on the natural substrate camphor. As is the case for camphor, the P450cam exhibits stereoselectivity for binding (R)- and (S)-2-ethylhexanol. Kinetic studies indicate (R)-2-ethylhexanoic acid is produced 3.5 times as fast as the (S)-enantiomer. In a racemic mixture of 2-ethylhexanol, P450cam produces 50% more (R)-2-ethylhexanoic acid than (S)-2-ethylhexanoic acid. The reason for stereoselective 2-ethylhexanoic acid production is seen in regioselectivity assays, where (R)-2-ethylhexanoic acid comprises 50% of total products while (S)-2-ethylhexanoic acid comprises only 13%. (R)- and (S)-2-ethylhexanol exhibit similar characteristics with respect to the amount of oxygen and reducing equivalents consumed, however, with (S)-2-ethylhexanol turnover producing more water than the (R)-enantiomer. Crystallographic studies of P450cam with (R)- or (S)-2-ethylhexanoic acid suggest that the (R)-enantiomer binds in a more ordered state. These results indicate that wild-type P450cam displays stereoselectivity toward 2-ethylhexanoic acid synthesis, providing a platform for rational active site design.

PubMed ID: 11583152

MeSH Terms: Camphor 5-Monooxygenase/isolation & purification; Camphor 5-Monooxygenase/metabolism*; Camphor/metabolism; Caproates/metabolism*; Computer Simulation*; Crystallography, X-Ray; Kinetics; Models, Molecular; Molecular Structure; Protein Structure, Tertiary*; Pseudomonas putida/enzymology