A natural heme‐signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse ω‐hydroxylation

Abstract

A novel naturally occurring heme-signature variant of CYP267A1 from myxobacterium Sorangium cellulosum So ce56 and its mutant L366F, the actual mimic of the 'conserved' heme-signature of cytochromes P450, were heterologously expressed in Escherichia coli in a soluble form and purified. The UV-visible characteristics of both variants were highly similar. Although leucine replaced the phenylalanine in the heme-signature domain of CYP267A1, EPR measurements of the ligand-free wild-type CYP267A1 and the mutant L366F showed low-spin rhombic species suggesting a conserved heme environment of the P450s. The need of primary redox partners for the orphan P450 was sustained by the bovine redox system and a class-I electron transfer path was provided during fatty acid hydroxylation. CYP267A1 showed higher activity and produced more diverse x-hydroxylated products compared with L366F. In both enzymes the regioselectivity of the fatty acid hydroxylation shifted towards the inner carbon atoms of the fatty acid chains with increasing carbon chain lengths. Our docking results in a homology model of the protein showed that longer fatty acids need to be folded to fit into the binding pocket. In the mutant L366F, the x-1 and x-2 positions which exhibit the largest electron density of the highest occupied molecular orbital are preferred. It is speculated that the leucine heme-signature variant of P450 might have evolved under selective evolutionary pressure, which confers an increased advantage to generate a broader spectrum of related alcohols and carboxylic acids required for the bacterial homeostasis or metabolism in a particular ecological niche.

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

“…For the past few years, we have been engaged in the study of novel P450s from myxobacteria, and some P450s from S. cellulosum So ce56 have shown a potential for industrial and biotechnological applications (Khatri et al, 2010a(Khatri et al, ,b, 2013(Khatri et al, , 2014Ly et al, 2012;Ringle et al, 2013). We were also able to develop an efficient E. coli-based whole-cell bioconversion system for some myxobacterial P450s .…”

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

“…Glucose dehydrogenase (GDH) from Bacillus megaterium (gdhIV; GenBank D10626) was expressed by pET-22b(+) in E. coli BL21(DE3) as described previously (Khatri et al, 2015).…”

“…After the desired reaction time, samples containing lauric acid (C12) 7 were prepared for GC/MS analysis by addition of 5 µl of 37% HCl and extracted twice with 500 µl diethyl ether (Khatri et al, 2015). All other target compounds were extracted with 200 µl ethyl acetate.…”

“…Analysis of C12 7 conversions and identification of conversion products was performed as described previously (Khatri et al, 2015). For analysis of target compounds 4-IPO 12, PK 13, 2-FPK 14, 2-HexF 16 and 2-HepF 17, the column temperature was maintained at 70°C for 2 min, ramped to 220°C with a rate of 10°C·min −1 , then to 300°C at a rate of 40°C·min −1 , and held at 300°C for 1 min.…”

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

“…[86] The dissociation constant (K d ) of (+)-eremophilene for CYP264B1 was calculated with SigmaPlot 9.0 (Systat Software, San Jose, CA) by fitting the peak-to-trough difference against the substrate concentration to the non-linear tight-binding quadratic Equation (1):…”