Bacterial P450 Research Articles

Redox Partners: Function Modulators of Bacterial P450 Enzymes

The superfamily of cytochrome P450 monooxygenases (P450s) is widespread in all kingdoms of life. Functionally versatile P450s are extensively involved in diverse anabolic and catabolic processes. P450s require electrons to be transferred by redox partners (RPs) for O2 activation and substrate monooxygenation. Unlike monotonic eukaryotic cytochrome P450 reductases, bacterial RP systems are more diverse and complicated. Recent studies have demonstrated that the type, the amount, the combination, and the mode of action of bacterial RPs can affect not only the catalytic rate and product distribution but also the type and selectivity of P450 reactions. These results are leading to a novel opinion that RPs not only function as auxiliary electron transfer proteins but are also important P450 function modulators.

Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity

AbstractThe evaluation of drug metabolites is compulsory during drug development. Since recently, bacterial cytochromes P450 and their mutated variants have attracted considerable interest as an alternative to hepatic P450s for the synthesis of human drug metabolites. Thus, straightforward screening approaches are required that enable rapid identification and evaluation of drug‐metabolizing bacterial P450s with different product selectivities. Herein, we report a two‐step screening method for discovery and characterization of new P450s from actinomycetes that enable oxidation of various drugs. In the first step, substrate profiling with three structurally different model drugs, ritonavir, testosterone, amitriptyline, allowed us to select CYP105D and CYP107Z from Streptomyces platensis DSM 40041 that accepted all model substrates and produced human‐like drug metabolites. In the second step, activity tests with an array of 25 structurally‐related molecules and derivatives of the three model compounds revealed a correlation between structural variations in the target drugs and the enzyme chemo‐ and regioselectivity.

Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1

Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2.

Highly selective bile acid hydroxylation by the multifunctional bacterial P450 monooxygenase CYP107D1 (OleP)

ObjectiveRegio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus.ResultsCo-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6β-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L−1 h−1. NMR spectroscopy confirmed the identity of MDCA as the sole product.ConclusionsThe multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.

Exploring the molecular basis for substrate specificity in homologous macrolide biosynthetic cytochromes P450

Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99-Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes.

Regioselective hydroxylation pathway of tenatoprazole to produce human metabolites by Bacillus megaterium CYP102A1

Abstract Tenatoprazole, a proton pump inhibitor drug candidate, is developed as an acid inhibitor to treat gastric acid hypersecretion disorders, such as gastric ulcer and reflux esophagitis. Tenatoprazole is known to be metabolized to three major metabolites—tenatoprazole sulfone, 5’-hydroxylated metabolite, and tenatoprazole sulfide—in human livers mainly by CYP2C19 and CYP3A4. In this study, an enzymatic strategy for the production of human metabolites of tenatoprazole was developed using bacterial P450 enzymes. A set of CYP102A1 mutants catalyzed the regioselective hydroxylation reactions of tenatoprazole. The major product of tenatoprazole by CYP102A1 is 5’-OH tenatoprazole, a major human metabolite produced by human CYP2C19 and CYP3A4. As another major metabolite of tenatoprazole, tenatoprazole sulfide is formed via a non-enzymatic conversion without a P450 system. In addition, 5’-OH tenatoprazole sulfide was found as a minor metabolite, which can be formed via a 5’-hydroxylation reaction of tenatoprazole sulfide by CYP102A1 and/or a non-enzymatic reduction of 5’-OH tenatoprazole to a sulfide form. Chemical synthesis of the 5’-OH tenatoprazole is not currently possible. In conclusion, an enzymatic synthesis of 5’-OH tenatoprazole, a major human metabolite of tenatoprazole, was developed by using mutants of CYP102A1 from Bacillus megaterium as a biocatalyst and tenatoprazole as a substrate.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.)

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as “blooms”. The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe–4S] cluster containing ferredoxins (eight), a single [2Fe–2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA-assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1-fold higher than that of the S. solfataricus PCNA-assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.

Construction and functional analysis of a whole-cell biocatalyst based on CYP108N7.

Cytochrome P450 enzymes are versatile biocatalysts with great potential in biotechnology. A new bacterial P450 was identified from the genome of Rhodococcus wratislaviensis NBRC 100605 and annotated as CYP108N7. The enzyme accepted the ferredoxin and ferredoxin reductase from spinach as surrogate redox partners for improved electron transfer efficiency. It was heterologous expressed in Escherichia coli together with the redox partners and a glucose dehydrogenase which supplied the reduced cofactor NADPH. The resulting whole-cell biocatalyst catalyzed a variety of reactions including sulfoxidation, epoxidation, hydroxylation, demethylation and dehalogenation. Remarkable stereoselectivity was observed in asymmetric sulfoxidation reaction, which could deliver chiral sulfoxides with >99% ee from thioanisole and derivatives.

Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469) from Streptomyces avermitilis.

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The Km and kcat values for compactin were 39.1 ± 8.8 µM and 1.12 ± 0.09 min-1, respectively. The kcat/Km value for compactin (0.029 min-1·µM-1) was lower than that for diclofenac (0.114 min-1·µM-1). Spectroscopic analysis showed that CYP105D7 binds to compactin with a Kd value of 17.5 ± 3.6 µM. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

AbstractBacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support.

Bacterial cytochrome P450s (P450s), which catalyze regio- and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost-effective industrial manufacturing. However, immobilization of P450s is difficult because electron-transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein-mediated co-immobilization of a bacterial P450, and its electron-transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co-immobilized enzymes catalyzed monooxygenation because the electron-transfer protein fused to PCNA via a single peptide linker retained its electron-transport function.

Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel

Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking “proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins” (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD+. The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions.

Enzymatic C(sp3)-H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones.

Cytochrome P450 enzymes can effectively promote the activation and cyclization of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C–H insertion reaction. Investigation of the substrate scope shows that while benzylic/allylic C–H bonds are most readily aminated by these biocatalysts, stronger, secondary C–H bonds are also accessible to functionalization. Leveraging this “non-native” reactivity and assisted by fingerprint-based predictions, improved active-site variants of the bacterial P450 CYP102A1 could be identified to mediate the aminofunctionalization of two terpene natural products with high regio- and stereoselectivity. Mechanistic studies and KIE experiments show that the C–H activation step in these reactions is rate-limiting and proceeds in a stepwise manner, namely, via hydrogen atom abstraction followed by radical recombination. This study expands the reactivity scope of P450-based catalysts in the context of nitrene transfer transformations and provides first-time insights into the mechanism of P450-catalyzed C–H amination reactions.

Cytochrome P450cin (CYP176A1).

Cytochrome P450cin (P450cin) (CYP176A1) is a bacterial P450 enzyme that catalyses the enantiospecific hydroxylation of 1,8-cineole to (1R)-6β-hydroxycineole when reconstituted with its natural reduction-oxidation (redox) partner cindoxin, E. coli flavodoxin reductase, and NADPH as a source of electrons. This catalytic system has become a useful tool in the study of P450s as not only can large quantities of P450cin be prepared and rates of oxidation up to 1,500min(-1) achieved, but it also displays a number of unusual characteristics. These include an asparagine residue in P450cin that has been found in place of the usual conserved threonine residue observed in most P450s. In general, this conserved threonine controls oxygen activation to create the potent ferryl (Fe(IV=O) porphyrin cation radical required for substrate oxidation. Another atypical characteristic of P450cin is that it utilises an FMN-containing redoxin (cindoxin) rather than a ferridoxin as is usually observed with other bacterial P450s (e.g. P450cam). This chapter will review what is currently known about P450cin and how this enzyme has provided a greater understanding of P450s in general.

Use of chemical auxiliaries to control p450 enzymes for predictable oxidations at unactivated C-h bonds of substrates.

Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio- and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drug-metabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.

Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 μM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family.

Human NAD(P)H:quinone Oxidoreductase 1 (NQO1)-Mediated Inactivation of Reactive Quinoneimine Metabolites of Diclofenac and Mefenamic Acid

quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites.

Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450

BackgroundCytochrome P450 monooxygenases – able to regio- and stereoselectively hydroxylate non-activated carbon atoms – are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16α-hydroxytestosterone. Since the hydroxylation at 16α-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids.ResultsCYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16α-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 μmol substrate consumed min-1 μmol-1 CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 μmol substrate consumed min-1 μmol-1 CYP154C5.ConclusionCYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16α-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (μmol substrate consumed μmol-1 CYP154C5) exceeding 2000.