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

This study examines the ability of P450cam. to catalyze the formation of 2- ethylhexanoic acid from 2-ethylhexanol relative to its activity on the natu ral substrate camphor. As is the case for camphor, the P450cam exhibits ste reoselectivity for binding (R)- and (S)-2-ethylhexanol. Kinetic studies ind icate (R)-2-ethylhexanoic acid is produced 3.5 times as fast as the (S)-ena ntiomer. In a racemic mixture of 2-ethylhexanol, P450cam produces 50% more (R)-2-ethylhexanoic acid than (S)-2-ethylhexanoic acid. The reason for ster eoselective 2-ethylhexanoic acid production is seen in regioselectivity ass ays, where (R)-2-ethylhexanoic acid comprises 50% of total products while ( S)-2-ethylhexanoic acid comprises only 13%. (R)- and (S)-2-ethylhexanol exh ibit similar characteristics with respect to the amount of oxygen and reduc ing equivalents consumed. however, with (S)-2-ethylhexanol turnover produci ng 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 dis plays stereoselectivity toward 2-ethylhexanoic acid synthesis, providing a platform for rational active site design.