Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

Abstract

cThe cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product ␥-hydroxy-N-methyl-L-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application. C yclosporine (CsA) (Fig. 1), a lipophilic cyclopeptide consisting of 11 amino acids, was first isolated from the soil fungus Tolypocladium inflatum (1). It is a well-known immunosuppressant drug that has been widely used in organ transplantation to prevent allograft rejection and in the treatment of autoimmune diseases (2, 3). Despite its immunosuppressant activity, CsA provokes several side effects, such as nephrotoxicity, hypertension, and hirsutism (4, 5). Among these side effects, the strong hairgrowth-stimulating activity of CsA has attracted significant attention from both the academic community and the cosmetics industry due to its great potential to treat alopecia (6, 7). Mechanistically, it was revealed that CsA stimulates the growth of both murine hair epithelial cells and epidermal keratinocytes at an optimum concentration through inhibiting the expression and translocation of some protein kinase C isozymes that act as negative hair-growing factors (8).Unfortunately, CsA was not practical for use as a hair-restoring agent due to its strong immunosuppressive activity. To decouple these two activities, a wide spectrum of CsA derivatives with alternative amino acid substitutions was prepared and evaluated (9), among which the derivative ␥-hydroxy-N-methyl-L-Leu4-CsA (CsA-4-OH) (Fig. 1) was found to have lost immunosuppressive activity while retaining the considerable hair-growth-promoting effect (10). Regarding preparation, the highly regioselective hydroxylation of CsA by a chemical catalyst is extremely challenging. Thus, thousands of actinomycete strains were screened ...

In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

“…The Hill equation can be used to indicate cooperative kinetics. Indeed, several studies have found that some P450s are able to fit into its active site with large molecules such as cyclosporine [ 60 ] or two substrates at once [ 57 ]. The two substrates can be the same or different molecules allowing the homotropic or heterotropic cooperative kinetics, respectively.…”

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

“…Notably, the protein pair SelFdx1499 (Fe 2 S 2 )/SelFdR0978 (plastidic-type FdR) from the cyanobacterial strain Synechococcus elongatus PCC 7942 has been shown to be an optimal combination for supporting in vitro reactions of prokaryotic P450s, including MycG, PikC, P450sca-2 and others (76). Besides the above-mentioned P450 reaction matrix network, the protein pair SelFdx1499/SelFdR0978 has also been shown to be optimal for the site-selective hydroxylation of CsA by CYP-sb21 and CYP-pa1 (45,47), the uncommon ester-to-ether transformation catalyzed by Rif16 in rifamycin biosynthesis (77), the tandem ether installation and hydroxylation by AmbV involved in neoabyssomicin/abyssomicin biosynthesis (78), and the biosynthesis of phenylserine (␤-OH-Phe) unit in atratumycin by Atr27 (79).…”

Low-Level Organic Solvents Improve Multienzyme Whole-Cell Catalytic Synthesis of Myricetin-7-O-Glucuronide

“…Given the attractive advantages of conventional water-miscible organic solvents, including their simple management, high solubility for various organic substances, and capacities to improve the enantio-selectivity and activity of biocatalysts [11,12], organic solvents have been used as "cosolvents" and "permeability enhancers" to stimulate substrate transfer across cells [7,10], but the choice and amount of added organic solvent are highly empirical and generally undesirable for large-scale applications [13]. In most single-enzyme whole-cell catalytic processes, dimethyl sulfoxide (DMSO) and methanol (MeOH) are commonly used as solubility enhancers to promote the biosynthesis of glycosides and hydrophobic compounds [14,15]. The use of a recombinant single enzyme overexpressed in Escherichia coli is employed as a whole-cell biocatalyst treated with 60% acetone (v/v) [16] or 10% ethanol (EtOH) [11] to enhance the permeabilization of cell membranes and, further, to promote high yields of L-phenylalanine and L-menthol.…”