A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann's ketone.

25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration.

Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX.

Allylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC-MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.

A novel P450-based biocatalyst for the selective production of chiral 2-alkanols.

A P450 monooxygenase from Nocardia farcinica (CYP154A8) catalyses the stereo- and regioselective hydroxylation of n-alkanes, still a challenging task in chemical catalysis. In a biphasic reaction system, the regioselectivity for the C2-position of C7-C9 alkanes was over 90%. The enzyme showed strict S-selectivity for all tested substrates, with enantiomeric excess (ee) of up to 91%.