Bacterial P450 Monooxygenase Research Articles

Riboflavin Is Directly Involved in N-Dealkylation Catalyzed by Bacterial Cytochrome P450 Monooxygenases.

Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals.

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.

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.

Biocatalytic Synthesis of Dihydroxynaphthoic Acids by Cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was found to exhibit oxidation activity towards three hydroxynaphthoic acids. Whole cells of the recombinant Escherichia coli strain expressing CYP199A2 efficiently catalyzed the regioselective oxidation of 1-, 3-, and 6-hydroxy-2-naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihydroxynaphthoic acid respectively. These results suggest that CYP199A2 might be a useful oxidation biocatalyst for the synthesis of dihydroxynaphthoic acids.

Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy.CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid,at a rate of 130 mol (mol P450)(-1) min(-1). Furthermore,CYP199A2 also oxidized quinoline-6-carboxylic acid,although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2,C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

Preparative use of isolated CYP102 monooxygenases—A critical appraisal

Isolated P450 monooxygenases have for long been neglected catalysts in enzyme technology. This is surprising as they display a remarkable substrate specificity catalyzing reactions, which represent a challenge for classic organic chemistry. On the other hand, many P450 monooxygenases are membrane bound, depend on rather complicated electron transfer systems and require expensive cofactors such as NAD(P)H. Their activities are low, and stability leaves much to be desired. The use of bacterial P450 monooxygenases from CYP102 family allows overcoming some of these handicaps. They are soluble and their turnovers are high, presumably because their N-terminal heme monooxygenase and their C-terminal diflavin reductase domain are covalently linked. In recent years, protein engineering approaches have been successfully used to turn CYP102 monooxgenases into powerful biocatalysts.

P450BM-3 and other Inducible Bacterial P450 Cytochromes: Biochemistry and Regulation

When I began this review my goal was to present a coherent overview of the biochemistry and regulation of the inducible P450 cytochromes of bacteria. Now, at the end, I wonder if a unified perspective is possible at this time. On the basis of admittedly limited data, bacterial P450 systems seem as different from each other as they are, as a group, from the mammalian P450 cytochromes. The most obvious physical difference between the bacterial monooxygenases and their mammalian counterparts is solubility; with several possible exceptions (69, 70, 76), bacterial P450s are soluble whereas the microsomal and mitochondrial P450s are membrane-associated proteins. In structure and organization, however, the few well-characterized prokaryotic P450-dependent systems vary widely. The three-component arrangement is probably most common but even here variation is apparent. The P450cam putidaredoxin reductase contains only FAD and is quite specific for NADH (35, 39); the P450meg megaredoxin reductase contains only FMN and is specific for NADPH (59, 60). Putitive two-component P450 systems in bacteria have not yet been adequately characterized but the P450oct and P450npd monooxygenases (69, 70, 93) could well be organized in this way. The catalytically self-sufficient P450BM-3 is currently the only single-component P450-dependent monooxygenase known but additional examples of this arrangement may well be found in other bacteria. Paradoxically, P450BM-3 is structurally much more analogous to liver microsomal P450 systems than to any other bacterial P450 monooxygenase characterized to date. Another generally recognized difference between prokaryotic and eukaryotic P450s pertains to function; most known bacterial P450-dependent systems initiate the oxidation of recalcitrant carbon compounds so that the hosts can utilize them as sole carbon sources for growth. Some lower eukaryotes [certain yeasts, for example (134)] also employ P450-dependent systems in this way but, among most fungi as well as in higher eukaryotes, P450 cytochromes are involved in specific pathways of sterol or other lipid syntheses or, as in the mammalian liver microsomal systems, in detoxification reactions.(ABSTRACT TRUNCATED AT 400 WORDS)