Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates >70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 Å for an indazole complex and 2.6 Å for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr 303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe 478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216 QXXNN 220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed -1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.Cytochrome P-450 (P-450) 3 is a superfamily of enzymes involved in monooxygenation of both endogenous and exogenous substrates. A subset of these enzymes, including cytochrome P-450 2E1 (CYP2E1), are known for their role in the clearance of drugs and other xenobiotics by introducing or unmasking sites for subsequent conjugation and elimination from the body. From a biochemical standpoint these enzymes are fascinating for the diversity of substrates each xenobioticmetabolizing P-450 can metabolize, yet often with exquisite selectivity in the metabolites generated. Unfortunately, in the process some P-450-mediated metabolism can produce toxic or carcinogenic products. Among cytochrome P-450 enzymes, CYP2E1 is particularly notable for this ability and the resulting toxicity (1). This activity is most substantial in the liver because CYP2E1 comprises over 50% of the hepatic cytochrome P-450 mRNA (2) and 7% of the hepatic cytochrome P-450 protein (3). However, CYP2E1 is also expressed at lower levels in a variety of extrahepatic tissues (4), where it is thought to play a role in the metabolism of important endogenous molecules. CYP2E1 levels and the resulting toxicity varies markedly in response to alcohol consumption (5), diabetes (6), obesity (7), and fasting (8). Thus, the action of CYP2E1 can have a significant influence on human health and drug metabolism.CYP2E1 has been connected with liver toxicity through two ...
Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both FAD and NADPH for activity, and was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics using an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a non-substrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA in relation to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (para-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady state experiments indicate that PvdA:FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to para-hydroxybenzoate hydroxylase (PHBH from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. P. aeruginosa is an opportunistic human pathogen that under iron-limiting conditions produces two siderophores, pyochelin and pyoverdin, which contribute to virulence (1,2). Pyoverdin, a hydroxamate siderophore, chelates iron with high affinity, and has the ability to remove iron from host proteins such as transferrin and lactoferrin (3). Derivatives of ornithine, both hydroxyornithine and formyl-hydroxyornithine, are incorporated into the peptide backbone of pyoverdin and directly coordinate the iron (4).Ornithine hydroxylase (PvdA or L-ornithine N 5 -oxygenase) is the first enzyme involved in the derivatization of the ornithine, hydroxylating the primary amine of the side chain ( Figure 1a). PvdA is part of the pyoverdin locus (5) and PvdA deletion mutants are pyoverdin-deficient (6, 7). PvdA is functionally related to the lysine hydroxylase (IucD) of E. coli, which is required for production of the aerobactin siderophore (Figure 1b), para-hydroxybenzoate hydroxylase (PHBH) of the soil bacterium P. fluorescens, which is important in the biodegradation of lignin from wood (Figure 1c), and flavin-containing monooxygenases (FMOs) from a variety of organisms from bacteria to mammals involved in xenobiotic detoxification (Figure 1d) . FAD is reduced by NADPH and can then donate electrons to molecular oxygen ( Figure 1). The end result is the production of H 2 O, and a hydroxyl group is added to the sidechain amine of lysine (IucD), the activated aromatic ring of the hydroxybenzoate (PHBH), or to a ...
The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and cosubstrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP ؉ remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the ornithine side chain amine using NADPH as the electron donor and molecular oxygen (1). As a microbial N-hydroxylating monooxygenase, PvdA is considered to be a member of the class B flavoprotein monooxygenases, which share the following characteristics. 1) FAD is a stably bound cofactor; 2) NADPH, but not NADH, serves as the electron-donating cosubstrate and remains bound during the oxidative half-reaction; 3) they are composed of FAD and NADPH dinucleotidebinding domains; and 4) they are encoded by a single gene (2). PvdA meets these requirements with some caveats: PvdA does not stably bind FAD (1), and no previous work documents structure or confirms NADP ϩ binding through the oxidative half-reaction.The known class B flavoprotein monooxygenases are divided into three subclasses (2). Microbial N-hydroxylating monooxygenases catalyze the hydroxylation of primary amines and include PvdA (1, 3, 4), the ornithine hydroxylase from Aspergillus fumigatus (SidA) (5, 6), and the lysine hydroxylases (7,8). A mechanism for PvdA (Fig. 1a) has been proposed previously (4). In short, NADPH reduces the oxidized flavin in the reductive half-reaction. In the oxidative half-reaction, ornithine binding accelerates the addition of oxygen to the flavin and makes a short-lived peroxyflavin intermediate (which is slow to form and long-lived in the absence of ornithine), followed by the hydroperoxyflavin intermediate. The hydroperoxyflavin donates the distal oxygen atom to the ornithine, forming hydroxyornithine and the hydroxyflavin intermediate. The hydroxyflavin intermediate dehydrates to regenerate the oxi...
PvdA catalyzes the hydroxylation of the sidechain primary amine of ornithine in the initial step of the biosynthesis of the Pseudomonas aeruginosa siderophore pyoverdin. The reaction requires FAD, NADPH, and O2. PvdA uses the same co-substrates as several flavin-dependent hydroxylases that differ one from another in the kinetic mechanisms of their oxidative and reductive half-reactions. Therefore, the mechanism of PvdA was determined by absorption stopped-flow experiments. By contrast to some flavin-dependent hydroxylases (notably, p-hydroxybenzoate hydroxylase), binding of the hydroxylation target is not required to trigger reduction of the flavin by NADPH: the reductive half-reaction is equally facile in the presence and absence of ornithine. Reaction of O2 with FADH2 in the oxidative half-reaction is accelerated by ornithine 80-fold, providing a mechanism by which PvdA can ensure coupling of NADPH and ornithine oxidation. In the presence of ornithine, the expected C(4a)-hydroperoxyflavin intermediate with 390-nm absorption accumulates and decays to the C(4a)-hydroxyflavin in a kinetically competent fashion. The slower oxidative half-reaction that occurs in the absence of ornithine involves accumulation of an oxygenated flavin species and two subsequent states that are tentatively assigned as C(4a)-peroxy- and -hydroperoxyflavin intermediates and the oxidized flavin. The enzyme generates stoichiometric hydrogen peroxide in lieu of hydroxyornithine. The data suggest that PvdA employs a kinetic mechanism that is a hybrid of those previously documented for other flavin-dependent hydroxylases.
Summary Human xenobiotic-metabolizing cytochrome P450 (P450) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally a single ligand can interact with multiple cytochrome P450 enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the P450 superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human cytochrome P450 enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key amino acids involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and a CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å), have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual amino acids lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine. Hyperlinking to databases The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) with the following codes: CYP2A6 with pilocarpine (3T3R), CYP2A6 I208S/I300F/G301A/S369G with pilocarpine (3T3Q), CYP2A13 with pilocarpine (3T3S), and CYP2E1 with pilocarpine (3T3Z).
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