Sylvie Kandel

Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone Jasmonoyl-isoleucine for catabolic turnover.

Background: Oxidized derivatives of the plant hormone jasmonoyl-isoleucine accumulate in wounded Arabidopsis leaves. Results: Cytochromes P450 CYP94C1 and CYP94B3 cooperate to catalyze the formation of 12OH-JA-Ile and 12COOH-JA-Ile. Conclusion: CYP94C1 and CYP94B3 define a major route for JA-Ile catabolism. Significance: Elucidation of CYP94-mediated JA-Ile oxidation opens new avenues for understanding jasmonate metabolism and signaling. The jasmonate hormonal pathway regulates important defensive and developmental processes in plants. Jasmonoyl-isoleucine (JA-Ile) has been identified as a specific ligand binding the COI1-JAZ co-receptor to relieve repression of jasmonate responses. Two JA-Ile derivatives, 12OH-JA-Ile and 12COOH-JA-Ile, accumulate in wounded Arabidopsis leaves in a COI1- and JAR1-dependent manner and reflect catabolic turnover of the hormone. Here we report the biochemical and genetic characterization of two wound-inducible cytochromes P450, CYP94C1 and CYP94B3, that are involved in JA-Ile oxidation. Both enzymes expressed in yeast catalyze two successive oxidation steps of JA-Ile with distinct characteristics. CYP94B3 performed efficiently the initial hydroxylation of JA-Ile to 12OH-JA-Ile, with little conversion to 12COOH-JA-Ile, whereas CYP94C1 catalyzed preferentially carboxy-derivative formation. Metabolic analysis of loss- and gain-of-function plant lines were consistent with in vitro enzymatic properties. cyp94b3 mutants were largely impaired in 12OH-JA-Ile levels upon wounding and to a lesser extent in 12COOH-JA-Ile levels. In contrast, cyp94c1 plants showed wild-type 12OH-JA-Ile accumulation but lost about 60% 12COOH-JA-Ile. cyp94b3cyp94c1 double mutants hyperaccumulated JA-Ile with near abolition of 12COOH-JA-Ile. Distinct JA-Ile oxidation patterns in different plant genotypes were correlated with specific JA-responsive transcript profiles, indicating that JA-Ile oxidation status affects signaling. Interestingly, exaggerated JA-Ile levels were associated with JAZ repressor hyperinduction but did not enhance durably defense gene induction, revealing a novel negative feedback signaling loop. Finally, interfering with CYP94 gene expression affected root growth sensitivity to exogenous jasmonic acid. These results identify CYP94B3/C1-mediated oxidation as a major catabolic route for turning over the JA-Ile hormone.

Cytochrome P450-dependent fatty acid hydroxylases in plants

In plants, hydroxy-fatty acid production is mainly the result of enzymatic reactions catalyzed by cytochrome P450 dependent fatty acid hydroxylases. One can distinguish ω-hydroxylases that catalyze the hydroxylation of the terminal methyl of aliphatics acids (ω position) and sub-terminal or in-chain hydroxylases that oxidize carbons in the chain (ω-n position). Since both types of enzymes were discovered about three decades ago, the majority of investigations have focused on the CYP94 and CYP86 families, which mediate ω-hydroxylations. The activities of ω-hydroxylases in cutin synthesis have been clearly established, but the studies of LCR (LACERATA) and att1 (aberrant induction of type three genes), which are the first Arabidopsis thaliana mutants with alterations in coding sequences of CYP86A8 and CYP86A2, show that these types of ω-hydroxylases can be involved in many aspects of plant development. The existence of different ω-hydroxylases in plants with distinct regulation patterns suggests that these enzymes mediate diverse biological processes. Much less information concerning in-chain hydroxylases is available despite the fact that they were initially reported along with ω-hydroxylases. This lack of information might be explained by the very few examples of sub-terminal hydroxy-fatty acids described in plants. We present here the best characterized fatty acid hydroxylases and we discuss their possible roles in plant defense and development, fatty acid catabolism, plant reproduction and detoxification.

Characterization of a methyl jasmonate and wounding‐responsive cytochrome P450 of Arabidopsis thaliana catalyzing dicarboxylic fatty acid formation in vitro

A fatty‐acid‐metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12‐hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12‐hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in‐chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16× higher in microsomes from 24‐h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12‐, 11‐ and 10‐hydroxylauric acids, revealing for the first time the presence of fatty acid in‐chain hydroxylase in Arabidopsis.

Role of protein-protein interactions in cytochrome P450-mediated drug metabolism and toxicity.

Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes.

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

Background: CYP119 structural rearrangements were examined by 1H,15N HSQC NMR of 15N-labeled Phe residues and x-ray crystallography. Results: In solution, an open conformation of CYP119 is favored, but ligand-dependent F-G loop rearrangements produce two distinct closed conformations. Conclusion: The two closed conformations are generated by ligands that differ by a fluoro to chloro substitution. Significance: Cytochrome P450 enzymes ratchet among a discontinuous set of discrete conformations. Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development.

The cytochrome P450 (CYP) enzymes are a versatile superfamily of heme-containing monooxygenases, perhaps best known for their role in the oxidation of xenobiotic compounds. However, due to their unique oxidative chemistry, CYPs are also important in natural product drug discovery and in the generation of active metabolites with unique therapeutic properties. New tools for the analysis and production of CYP metabolites, including microscale analytical technologies and combinatorial biosynthesis, are providing medicinal chemists with the opportunity to use CYPs as a novel platform for lead discovery and development. In this review, we will highlight some of the recent examples of drug leads identified from CYP metabolites and the exciting possibilities of using CYPs as catalysts for future drug discovery.

Digging Deeper into CYP3A Testosterone Metabolism: Kinetic, Regioselectivity, and Stereoselectivity Differences between CYP3A4/5 and CYP3A7

The metabolism of testosterone to 6β-hydroxytestosterone (6β-OH-T) is a commonly used assay to evaluate human CYP3A enzyme activities. However, previous reports have indicated that CYP3A7 also produces 2α-hydroxytestosterone (2α-OH-T) and that a 2α-OH-T/6β-OH-T ratio may be a unique endogenous biomarker of the activity of the enzyme. Until now, the full metabolite and kinetic profile for testosterone hydroxylation by CYP3A7 has not been fully examined. To this end, we performed a complete kinetic analysis of the 6β-OH-T, 2α-OH-T, and 2β-hydroxytestosterone metabolites for recombinant Supersome CYP3A4, CYP3A5, and CYP3A7 enzymes and monitored metabolism in fetal and adult human liver microsomes for comparison. In general, a decrease in the velocity of the reaction was observed between CYP3A4 and the two other enzymes, with CYP3A7 showing the lowest metabolic capacity. Interestingly, we found that the 2α-OH-T/6β-OH-T ratio varied with substrate concentration when testosterone was incubated with CYP3A7, suggesting that this ratio would likely not function well as a biomarker for CYP3A7 activity. In silico docking studies revealed at least two different binding modes for testosterone between CYP3A4 and CYP3A7. In CYP3A4, the most energetically favorable docking mode places testosterone in a position with the methyl groups directed toward the heme iron, which is more favorable for oxidation at C6β, whereas for CYP3A7 the testosterone methyl groups are positioned away from the heme, which is more favorable for an oxidation event at C2α. In conclusion, our data indicate an alternative binding mode for testosterone in CYP3A7 that favors the 2α-hydroxylation, suggesting significant structural differences in its active site compared with CYP3A4/5.

Modulation of O-GlcNAc levels in the liver impacts acetaminophen-induced liver injury by affecting protein adduct formation and glutathione synthesis

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Variability in Potency Among Commercial Preparations of Berberine

ABSTRACT Berberine is an isoquinoline alkaloid plant extract that is widely available as a dietary supplement in the United States and has demonstrated efficacy in the treatment of type 2 diabetes mellitus and dyslipidemia. Because of its increased use and purported pharmacological properties, potential variations in product quality could pose a barrier to berberines safety and effectiveness in clinical practice. Thus, this study evaluated the potency of dietary supplements containing berberine available in the U.S. commercial market. Fifteen unique dietary supplements containing berberine were purchased through U.S. dietary supplement vendors. For each product, berberine was extracted from 3 unique capsules and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry. Percentage content based on the product label claim was determined for each product. The average berberine content across the products was found to be 75% ± 25% of the product label claim, with product potency ranging from 33% to 100%. Nine of the 15 tested products (60%) failed to meet the potency standards of 90% to 110% of labeled content claim, as commonly required of pharmaceutical preparations by the U.S. Pharmacopeial Convention. Evaluation of the relationship between product cost and the measured potency failed to demonstrate an association between quality and cost. Variability in product quality may significantly contribute to inconsistencies in the safety and effectiveness of berberine. In addition, the quality of the berberine product cannot be inferred from its cost.