Alkanes and Alicyclics

Engineering P450 enzymes into an alkane hydroxylase is an area of ongoing interest. A methane-oxidizing P450 enzyme may have important applications in the energy and chemicals sector while terminal oxidation of medium and long-chain alkanes and alkenes leads to fine chemicals. The CYP102 family of medium-chain fatty acid hydroxylases is a natural choice for engineering for alkane oxidation. Wild-type P450BM-3 showed low activity for octane oxidation (80 min-1). The A74G/F87V/L188Q mutant oxidized octane with fast NADPH

turnover (1760 min-1) and 40% coupling mainly to 3- and 4-octanol, with 17% 2-octanol [65]. Almost all P450BM-3 mutants reported to date produce secondary alcohols. Only Peters et al. have reported a variant containing 15 mutations that produces 10% 1-octanol [66].

Wild-type CYP102A3 shares 64% homology to the monooxygenase domain of P450BM-3 and shows limited regioselectivity for octane oxidation. However, CY-P102A3 has a more hydrophobic and narrower binding site than P450BM-3 and higher activity towards octane (110 min-1). The regioselectivity of CYP102A3 was changed by combination of error-prone PCR and site-directed mutagenesis to hydroxylate substrates not only at different subterminal carbons but also to a high extent at the terminal carbon. To enable high-throughput screening, a specific assay was developed, based on yeast alcohol dehydrogenase that is capable of discriminating between products of terminal and subterminal hydroxylation. Two CYP102A3 mutants, F88V/S189Q/A328V and S189Q/A328V, were identified and these produced 17% and 50% 1-octanol, respectively [67]. All three mutations occur in close proximity to the heme center and hinder approach of the substrate molecule to the heme, leading to terminal oxidation but which might also cause the poor coupling efficiencies of 5-7%.

By screening for activity with a p-nitrophenoxyoctane oxidation assay, a derivative of a p-nitrophenoxyoctanoate oxidation assay [19], P450BM-3 was taken through multiple rounds of directed evolution to identify the mutant "139-3" which oxidized octane with high turnover rates and 66% selectivity for 2-octanol [68]. This mutant was also capable of oxidizing alkanes as short as butane and propane. Further rounds of directed evolution combined with designed mutations in the active site lead to activity improvements and significant changes in regio-and stereoselectivity [66]. A mutant labeled "1-12G" yielded 82% 2-octanol, and several variants yielded small quantities of terminal hydroxylation products for a range of alkanes. An exciting development was the mutant 35-E11 prepared from the 9-10A by directed evolution while deliberately constricting the active site [69]. This mutant functions as an ethane hydroxylase with an ethanol formation rate of 0.4 min-1 and 0.8% coupling. High selectivity was observed in the epoxidation of terminal alkenes using a rationally designed variant of mutant 9-10A involving active site substitutions such as F87V and I263A [11].

P450cam has been redesigned for alkane oxidation. The size of the substrate and its shape in relation to the active site were crucial considerations. The alkane oxidation activity of the wild-type and Y96F mutants was highest for pentane/hexane and decreased steadily to octane [70]. Smaller substrate pockets generated by bulky substitutions such as V247L favored a sterically less demanding substrate such as hexane over 3-methylpentane [71]. Increases in activity towards smaller alkanes were achieved by introducing bulky residues high up in the active site to oblige the substrate to bind closer to the heme iron. The F87W/Y96F/T101L/ V247L mutant oxidized butane with a turnover rate of 750 min-1 compared with 0.4 min-1 for the wild-type enzyme [72]. Further bulky substitutions, together with the L358P mutation first used by Morishima and co-workers, were also added. The L358P mutation tightened the active site, pushed the heme towards the substrate, and increased the donor strength of the proximal cysteine thiolate by eliminating a hydrogen bond between Leu358 and Cys357 which promoted O-O bond cleavage and reduced uncoupling [73, 74]. The F87W/Y96F/T101L/T185M/L244M/V247L/ G248A/L358P mutant oxidized propane at 500 min-1 with 86% coupling. The NADH turnover rate for ethane was even higher (~800 min-1) but the coupling for ethanol formation was low (10.5%) [75].

Enantioselective hydroxylation of achiral substrates to chiral alcohols is an attractive route to fine chemicals and synthetic intermediates. P450cam was engineered to oxidize phenylcyclohexane selectively at C2, C3, or C4 of the cyclohexane ring [54, 76-78]. The Y96F mutant gave 81% cis-3-phenylcyclohexanol with 34% ee and the Y96F/V247A mutant gave 97% with 42% ee, while the Y96F/V247L mutant gave 83% trans-4-phenylcyclohexanol. Oxidation of a cyclopentanecarbox-ylic acid derivative by P450BM-3 mutants has been reported. The 139-3 mutant gave principally the S,S product while 1-12G formed the R,R diastereoisomer [79]. 2-Ethylhexanol is oxidized to 2-ethylhexanoic acid by P450cam [80, 81]. The F87W and Y96W mutants gave the acid exclusively, while the T185F mutation improved coupling but was less selective. The crystal structure of the wild type with the hexanol substrate bound revealed that the R-isomer was bound in a more ordered state and was the preferred substrate over the S-isomer.

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