4075-14-3

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp. W2061 (StCYP154C4-1) and Streptomyces sp. ATCC 11861 (StCYP154C4-2). Activity assays showed that both StCYP154C4-1 and StCYP154C4-2 can produce 2′-hydroxylated testosterone, which differs from the activity of a previously described NfCYP154C5 from Nocardia farcinica in terms of its 16α-hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP154C4 proteins, crystal structures of the ligand-unbound form of StCYP154C4-1 and the testosterone-bound form of StCYP154C4-2 were determined. Comparison with the previously determined NfCYP154C5 structure revealed differences in the substrate-binding residues, suggesting a likely explanation for the different patterns of testosterone hydroxylation, despite the high sequence similarities between the enzymes (54% identity). These findings provide valuable insights that will enable protein engineering for the development of artificial steroid-related CYPs exhibiting different regiospecificity.

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The regio- (and stereo-)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (ΔΔGbind) of the catalytically active substrate-binding poses, and the probability of the substrate to adopt a transition-state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450-catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ΔΔGbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.

There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism. 400 cytochrome P450 2D6 (CYP2D6) variants representing all possible amino acid exchanges at two important enzyme's residues were expressed and individually analyzed to investigate their influence on regioselective steroid hydroxylation. Steroids represent a substrate class atypical for wildtype CYP2D6. Employing this strategy CYP2D6 variants with improved activity and variants with altered region-preference were identified and characterized.

Summary: Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C - H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C - H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96 - 97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

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The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronunclear multiple bond correlation (HMBC) using broadband adiabetic 13C pulses and phase-sensitive data presentation. Testosterone, and rostenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the β-face of the steroids, at positions 2, 12, and 15. Copyright

The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3α-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3α-hydroxylase and converts AFB1 predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB1 regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB1 detoxification via 3α-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB1 oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11,265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB1 3α-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB1 molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB1 binding at the active site in either orientation leading to 3α- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB1 3α-hydroxylation and exo-8,9-oxidation.

Facile regiospecific syntheses of 2α-hydroxytestosterone (1) and 2β-hydroxytestosterone (2), starting from testosterone, are described.

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Bisdesmosides of two new furostanols (named furometagenin and furometanarthogenin) were isolated as their 22-O-methylated peracetates (designated as NF-1 and NF-2) from the subterranean part of Metanarthecium luteo-viride MAXIM. (Liliaceae).The structures of NF-1 and NF-2 have been elucidated as 11-O-α-L-arabinopyransyl-26-O-β-D-glucopyranosyl-22-O-methyl-furometagenin nonoacetate (12) and 11-O-α-L-arabinopyranosyl-26-O-β-D-glucopyranosyl-22-O-methyl-furometanarthogenin octaacetate (15) on the basis of chemical and physicochemical evidence.The 2β-acetoxy-4-en-3-one moiety included in the steroidal part of 15 has been demonstrated to undergo air oxidation during alkaline saponi fication followed by acidic hydrolysis in methanol to give a new sapogenol: 2-methoxy-11α-hydroxy-25R-spirosta-1,4-dien-3-one (2-O-methyl-dehydrometanarthogenin) (23).This secondary conversion in the A-ring of sapogenol part has been corroborated by the examination of the model steroids: 2β- and 2α-acetoxy-17β-chloroacetylandrost-4-en-3-ones (25, 28).Keywords: Metanarthecium luteo-viride; furostanol bisdesmoside; arabinosyl-glucosyl-furometagenin; arabinosyl-glucosyl-furometanarthogenin; furometagenin; furometanarthogenin; 2β-acetoxy-4-en-3-one steroid; air-oxidation; 2-O-methyl-dehydrometanarthogenin; CD