Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

Abstract

Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP + -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD + -dependent FDH was used.Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…No sigmoidal behavior has been reported for fatty acid binding or oxidation by P450 BM3 (Gustafsson et al, 2004), though isotope effect studies suggested that both laurate and palmitate could be simultaneously present in the active site (Rock et al, 2003). On the other hand, both homotropic and heterotropic cooperativity have been reported for the oxidation of non-natural substrates such as indole by P450 BM3 (Li et al, 2005;Maurer et al, 2005;Huang et al, 2007), and for drug metabolism by the R47L/ F87V/L188Q mutant in which allosteric effects were also proposed (van Vugt-Lussenburg et al, 2006).…”

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

“…-4.2 (Liang et al 2007) P. stipitis XR 1K8C (h) K270R, N272D 2.9 9.3 -1.2 (Maddock et al 2015) E. coli CaADH 1KEV (h) G198D, S199V, P201E, Y218A >1 ? -4.6 (Maurer et al 2005) B Log Relative Activity c (Medina et al 2001) A. PCC7119 FDNR 2BSA S223D 0.12 8.1x10 3 -5.4 (Nakanishi et al 1997) M. musculus CR 1CYD T38D 31 1.3x10 3 -0.51 (Paladini et al 2009 (Schepens et al 2000) S. bicolor MDH 7MDH (1CIV) G84D, S85I, R87Q, S88A 12 2.1x10 4 -0.96 (Scrutton et al 1990) E. coli GTR 1GET A179G, A183G, V197E, R198M, K199F, H200D, R204P 8.1 1.8x10 4 -1.5 (Shiraishi et al 1998) N. crassa CbR S920D, R932S 65 7.2x10 4 -2.6 (Takase et al 2014) S. sp. A1-R 3AFN H37N, G38S, R39H, K40V, A41D >1 ?…”

Hydroxylation of Dodecanoic Acid and (2R,4R,6R,8R)‐Tetramethyldecanol on a Preparative Scale using an NADH‐ Dependent CYP102A1 Mutant

“…[15] In our earlier publication we described processes for oxidation of alkanes in a biphasic system, where the electron supply from NADPH was supported by cofactor recycling with formate dehydrogenase. [16] Based on the knowledge gained, we have developed a process for oxidation of the highly branched fatty alcohol (2R,4R,6R,8R)-tetramethyldecanol (4) on a preparative scale. The presented reaction system is homogeneous, does not need any organic solvent and is therefore particularly benign to the environment.…”

“…[16] Although CYP102A1 3mDS showed a 4-fold lower NADH oxidation activity during the hydroxylation of (2R,4R,6R,8R)-tetramethyldecanol (4) (462 min…”

“…It has been proven previously that addition of both resulted in higher hydroxylation activity of a CYP102A1 mutant for an extended period of time. [16] For cofactor recycling the NAD + -dependent formate dehydrogenase (FDH) from Pseudomonas sp. 101 was used (Scheme 2).…”