Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine.Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.

The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated steroid hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel steroid hydroxylation positions.

Due to their bioactive properties as well as their application as precursors in chemical synthesis, hydroxylated isoprenoids and norisoprenoids are very valuable compounds. The efficient hydroxylation of such compounds remains a challenge in organic chemistry caused by the formation of a variety of side products and lack of overall regio- and stereoselectivity. In contrast, cytochromes P450 are known for their selective oxidation under mild conditions. Here, we demonstrate for the first time the ability of myxobacterial CYP260B1 and CYP267B1 from Sorangium cellulosum So ce56 to oxidize such carotenoid-derived aroma compounds. A focused library of 14 substrates such as ionones, damascones, as well as some of their isomers and derivatives was screened in vitro. Both P450s were capable of an efficient oxidation of all tested compounds. CYP260B1-dependent conversions mainly formed multiple products, whereas conversions by CYP267B1 resulted predominantly in a single product. To identify the main products by NMR spectroscopy, an Escherichia coli-based whole-cell system was used. CYP267B1 showed a hydroxylase activity towards the formation of allylic alcohols. Likewise, CYP260B1 performed the allylic hydroxylation of β-damascone [(E)-1-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-one] and δ-damascone [(E)-1-(2,6,6-trimethylcyclohex-3-enyl)but-2-en-1-one]. Moreover, CYP260B1 showed an epoxidase activity towards β-ionone [(E)-4-(2,6,6-trimethylcyclohex-1-enyl)but-3-en-2-one] as well as the methyl-substituted α-ionone derivatives raldeine [(E)-1-(2,6,6-trimethylcyclohex-2-enyl)pent-1-en-3-one] and isoraldeine [(E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-enyl)but-3-en-2-one]. In addition, to known products, also novel products such as 2-OH-δ-damascone [(E)-1-(5-hydroxy-2,6,6-trimethylcyclohex-3-enyl)but-2-en-1-one], 3-OH-allyl-α-ionone [(E)-1-(4-hydroxy-2,6,6-trimethylcyclohex-2-enyl)hepta-1,6-dien-3-one], and 4-OH-allyl-β-ionone [(E)-1-(3-hydroxy-2,6,6-trimethylcyclohex-1-enyl)hepta-1,6-dien-3-one] were identified during our studies.

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non-activated C-H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)-nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'β-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.