Exploring the Biocatalytic Potential of a Self‐Sufficient Cytochrome P450 from Thermothelomyces thermophila

Abstract

Among nature's arsenal of oxidative enzymes, cytochrome P450s (CYPs) catalyze the most challenging reactions, the hydroxylations of non-activated CÀ H bonds. Human CYPs are studied in drug development due to their physiological role at the forefront of metabolic detoxification, but their challenging handling makes them unsuitable for application. CYPs have a great potential for biocatalysis, but often lack appropriate features such as high and soluble expression, self-sufficient internal electron transport, high stability, and an engineerable substrate scope. We have probed these characteristics for a recently described CYP that originates from the thermophilic fungus Thermothelomyces thermophila (CYP505A30), a homolog of the well-known P450-BM3 from Bacillus megaterium. CYP505A30 is a natural monooxygenase-reductase fusion, is well expressed, and moderately tolerant towards temperature and solvent exposure. Although overall comparable, we found the stability of the enzyme's domains to be inverse to P450-BM3, with a more stable reductase compared to the heme domain. After analysis of a homology model, we created mutants of the enzyme based on literature data for P450-BM3. We then probed the enzyme variants in bioconversions using a panel of active pharmaceutical ingredients, and activities were detected for a number of structurally diverse compounds. Ibuprofen was biooxidized in a preparative scale whole cell bioconversion to 1-, 2-and 3hydroxyibuprofen.

Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7

“…Several studies have focused on the oxidation of capsaicin by P450s from mammals, fungi, and insects [15–18,30] . In humans, chemical modifications of the alkyl terminus by P450s greatly reduce the biological responses, likely due to decreased interactions between the metabolites and TRPV1 [33] .…”

“…The in vitro conversion of capsaicin by recombinant P450s (CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) can be used to investigate whether metabolites of capsaicin cause toxicity. Recently, self‐sufficient CYP505X from Aspergillus fumigatus and self‐sufficient CYP505A30 from Thermothelomyces thermophila were heterologously expressed in E. coli and found to catalyze the hydroxylation of capsaicin at the C8 position as well as the epoxidation of the double bond in the aliphatic chain [15–17] . Both ω‐1 hydroxylation and alkyl dehydrogenation have been suggested to be involved in the production of metabolites of capsaicin by insect P450s such as CYP6B6 and CYP9As [18] .…”

“…Capsaicin also has antitumor, anti‐obesity, and cardioprotective effects [14] . Several P450s have been reported to catalyze the oxidation of capsaicin, for example, the members of the CYP1 family, CYP2 family, CYP505 family, and the CYP9 family [15–18] …”

Structure of the fungal hydroxylase, CYP505A30, and rational transfer of mutation data from CYP102A1 to alter regioselectivity

“…Notable exceptions are CYP505E3 and CYP505D6 that also perform deeper in-chain hydroxylation of these substrates and CYP505B1 (Fum6), which is involved in fumonisin mycotoxin production. [15][16][17][18][19] Deviations from the typical sub-terminal hydroxylation pattern have also more recently been found in certain CYP102 members. BAMF2522 from Bacillus amyloliquefaciens DSM7 hydroxylates palmitic acid at positions ω-1 to ω-7.…”

Techniques for Enzyme Purification

“…Recently, Fraaije and coworkers used the IMAC technology for the purification of a selfsufficient cytochrome P450 monooxygenase from Thermothelomyces thermophila [86]. They also developed experimental protocols for screening of thermostable Baeyer-Villiger monooxygenases by purifying the His-tagged proteins with Ni-NTA beads in a 96-well plate format [87].…”