Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties

Abstract

The members of the CYP109 family (CYP109C1, CYP109C2, and CYP109D1) from Sorangium cellulosum So ce56 are among the 21 P450 enzymes, of which only CYP109D1 and CYP264B1 have so far been functionally characterized. Here, we attempted to characterize two other P450s (CYP109C1 and CYP109C2) for the first time and compare their biochemical, biophysical, and functional properties to those of the fatty acid hydroxylating CYP109D1. Considering the physiological importance of fatty acids, we investigated saturated fatty acid binding and conversion for all members of the CYP109 family. The interaction between the CYP109 members and different autologous/heterologous redox partners was compared using Biacore measurements in which only CYP109D1 and bovine adrenodoxin (Adx) formed a complex. Surprisingly, this interaction was similarly efficient as the interaction of Adx with its mammalian redox partners. The in vitro reconstitution assays showed no activity when using CYP109C1, although substrate binding was demonstrated; also, there was subterminal hydroxylation of saturated fatty acids, when using CYP109C2 and CYP109D1, where CYP109D1 was a much more efficient fatty acid hydroxylase. Interestingly, the hydroxylation position moved inside the fatty acid chain when using long-chain fatty acids, thus producing possible precursors for physiologically important products.

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

“…The EPR spectrum of ligand‐free CYP267A1 showed three rhombic low‐spin ( S = 1/2) ferric heme species with different contributions whose g ‐factors suggest a hexa‐coordinated heme iron with a cysteinate fifth ligand and the sixth aqua‐ligand. They correspond to the g ‐tensor elements of several bacterial P450s (Table ) and also to those that we have reported before for CYP109C1, CYP109C2 and CYP109D1 . In both cases, the equilibrium between high‐spin and low‐spin states appears to be completely shifted in favor of the low‐spin electronic state at low temperature, whereas at ambient temperature a shift to high‐spin configuration ( S = 5/2) was observed.…”

“…Spin‐state shifts upon substrate binding were assayed at room temperature under aerobic conditions using a UV–visible scanning photometer (UV‐2101PC; Shimadzu) equipped with two tandem cuvettes as described previously . The dissociation constants ( K d ) of C10–C13 for CYP267A1 and the L366F mutant were calculated by fitting the peak‐to‐trough difference against the substrate concentration to a nonlinear tight binding quadratic equation as described before .…”

“…Since our laboratory has more recently been engaged in the study of cytochromes P450 from the complement (CYPome) of the spore‐forming myxobacterium Sorangium cellulosum So ce56 , we have noticed a naturally occurring heme‐signature variant of CYP267A1, in which the conserved phenylalanine is replaced by leucine (L) in the conserved Cys‐pocket FxxGx(H/R)xCxG. Although the variation LxxGx(H/R)xCxG was also observed in CYP176A (P450cin) from Citrobacter braakii , a detailed biochemical and biophysical characterization has not been performed so far and the implication of this naturally acquired heme‐signature variant was not studied.…”

Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

“…). Interestingly, CYP109Q5 showed only minimal activity with fatty acids, which distinguishes this enzyme from the CYP109 members that are actually declared as fatty acid hydroxylases, such as CYP109B1, CYP109C1, CYP109C2 or CYP109D1 (Girhard et al ., ; Khatri et al ., ). The amino acid sequence identity of this novel C. apiculatus enzyme is 39% to CYP109B1, 44% to CYP109C1, 43% to CYP109C2 and 38% to CYP109D1.…”

Structure–function analysis for the hydroxylation of Δ4 C21‐steroids by the myxobacterial CYP260B1

“…We found that the strain Sorangium cellulosum So ce56 contains 21 P450s, 8 ferredoxins, and 2 ferredoxin reductases . However, only seven of the P450s, belonging to families CYP109 (CYP109C1, CYP109C2, and CYP109D1) , CYP264 (CYP264A1 and CYP264B1) , and CYP267 (CYP267A1 and CYP267B1) , were well characterized either for potential physiological or biotechnological applications. They are involved in the hydroxylation of fatty acids, terpenes, and terpenoids, as well as of drugs and drug derivatives .…”