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This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have never lost the enthusiasm for research that I learned in the summer of 1968 with Harry Broquist, and I have tried to instill this in the many trainees I have worked with. A sentence I use on closing slides is "It's not just a laboratory-it's a fraternity.", Competing Interests: Conflicts of interests The author declares that he has no conflict of interest with the contents of this article., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

As a result of technical advances in recombinant DNA technology and nucleotide sequencing, entire genome sequences have become available in the past decade and offer potential in understanding diseases. However, a central problem in the biochemical sciences is that the functions of only a fraction of the genes/proteins are known, and this is also an issue in pharmacology. This review is focused on issues related to the functions of cytochrome P450 (P450) enzymes. P450 functions can be categorized in several groups: 1) Some P450s have critical roles in the metabolism of endogenous substrates (e.g., sterols and fat-soluble vitamins). 2) Some P450s are not generally critical to normal physiology but function in relatively nonselective protection from the many xenobiotic chemicals to which mammals (including humans) are exposed in their diets [as well as more anthropomorphic chemicals (e.g., drugs, pesticides)]. 3) Some P450s have not been extensively studied and are termed "orphans" here. With regard to elucidation of any physiological functions of the orphan P450s, the major subject of this review, it is clear that simple trial-and-error approaches with individual substrate candidates will not be very productive in addressing questions about function. A series of liquid chromatography/mass spectrometry/informatics approaches are discussed, along with some successes with both human and bacterial P450s. Current information on what are still considered "orphan" P450s is presented. The potential for application of some of these approaches to other enzyme systems is also discussed.

The biochemical facets of toxicology have always had a major role in providing insight into mechanisms. Some of the history of the development of this area is summarized, including metabolism, enzymology, and the chemistry of reactive intermediates. Knowledge in these fields has had a major impact in the areas of drug metabolism and safety assessment, which are both critical steps in the development of pharmaceuticals and the rational use of commodity chemicals. The science of toxicology has developed considerably with input from other disciplines and today is poised to emerge as a predictive science with even more dramatic impact. The challenges ahead are considerable but there is renewed excitement in the potential of the field. As in the past, further advances in the field of toxicology will require the input of knowledge from many disciplines.

1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (Uvalues) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans. [ABSTRACT FROM AUTHOR]

This 11th Thematic Metals in Biology Thematic Series deals with copper, a transition metal with a prominent role in biochemistry. Copper is a very versatile element and both deficiencies and excesses can be problematic. The five Minireviews in this series deal with several aspects of copper homeostasis in microorganisms and mammals and the role of this metal in two enzymes, copper-only superoxide dismutase and cytochrome c oxidase. [ABSTRACT FROM AUTHOR]

P450s have attracted tremendous attention owing to not only their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely, the oxidation of unactivated C–H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s, including mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active-site features. Some of the substrate binding studies extending over a period of more than 40 years of dedicated work have been summarized in this review and categorized by the techniques employed in the binding studies. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes include a family of related enzymes that are involved in metabolism of vitamins, steroids, fatty acids, and other chemicals. This review presents a brief historical overview of the discovery and characterization of P450 enzymes extending from intermediary metabolism to the fields of drug metabolism and toxicology. Introductions to P450 enzyme structure and function are also presented. The goals of this review are to 1) provide an introduction to a few of the many aspects of P450 research relating to humans, 2) introduce additional ways of thinking about metabolism, 3) provide some basic examples of P450 enzymology, and 4) provide applications to topics widely taught in undergraduate courses in biochemistry. [ABSTRACT FROM AUTHOR]

From the x-ray crystal structure of CYP158A2 (Zhao, B., Guengerich, F. P., Bellamine, A., Lamb, D. C., Izumikawa, M., Lei, L., Podust, L. M., Sundaramoorthy, M., Reddy, L. M., Kelly, S. L., Kalaitzis, J. A., Stec, D., Voehler, M., Falck, J. R., Moore, B. S., Shimada, T., and Waterman, M. R. (2005) J. Biol. Chem. 280, 11599–11607), one of 18 cytochrome P450 (CYP) genes in the actinomycete Streptomyces coelicolor, ordered active site water molecules (WAT505, WAT600, and WAT640), and hydroxyl groups of the substrate flaviolin were proposed to participate in proton transfer and oxygen cleavage in this monooxygenase. To probe their roles in catalysis, we have studied the crystal structures of a substrate analogue (2-hydroxy-1,4-naphthoquinone) complex with ferric CYP158A2 (2.15 Å) and the flaviolin ferrous dioxygen-bound CYP 158A2 complex (1.8 Å). Catalytic activity toward 2-hydroxy-1,4-naphthoquinone was ∼70-fold less than with flaviolin. In the ferrous dioxygen-bound flaviolin complex, the three water molecules in the ferric flaviolin complex still occupy the same positions and form hydrogen bonds to the distal dioxygen atom. These findings suggest that CYP158A2 utilizes substrate hydroxyl groups to stabilize active site water and further assist in the iron-linked dioxygen activation. A continuous hydrogen-bonded water network connecting the active site to the protein surface (bulk solvent) not present in the other two ferrous dioxygen-bound P450 structures (CYP101A1/P450cam and CYP107A1/P450eryF) is proposed to participate in the proton-delivery cascade, leading to dioxygen bond scission. This ferrous-dioxygen structure suggests two classes of P450s based on the pathway of proton transfer, one using the highly conserved threonine in the I-helix (CYP101A1) and the other requiring hydroxyl groups of the substrate molecules either directly transferring protons (CYP107A1) or stabilizing a water pathway for proton transfer (CYP158A2). [ABSTRACT FROM AUTHOR]

Testosterone 6β-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6β-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6β-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-²H5; (ii) 6,6²H2; (iii) 6α-²H; (iv) 6³-²H; and (v) 6³-³H testosterone. Only the 6β-hydrogen was removed by P450 3A4 and not the 6α, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the β face. Analysis of the rates of hydroxylation of 6β-¹H-, 6β-²H-, and 6β-³H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect (Dk) of 15. The deuterium isotope effects on kcat and kcat/Km in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6β-²H substitution. The high Ok value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high Dk in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high Ok value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking. [ABSTRACT FROM AUTHOR]

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 Å) and flaviolin-bound (1.62 Å) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers. [ABSTRACT FROM AUTHOR]

Several issues regarding the rate-limiting nature of individual reaction steps in catalysis by rabbit liver cytochrome P450 (P450) 1A2 were addressed using anisoles and other substrates. Substrate binding is very fast (k > 106 M-1 s-1). Product release is not rate-limiting, as shown by the absence of bursts, placing rate-limiting steps at or before product formation. We had previously shown that the first 1-electron reduction step is fast (k > 700 min-1), even in the absence of ligand [Guengerich, F. P., and Johnson, W. W. (1997) Biochemistry 36, 14741-147500]. O2 binding to Fe2+rrous P450 is fast (k &e;= 106 M-1 s-1). The decay of the P450 Fe2+(2+)-substrate-O2 complex was slow in the absence of NADPH-P450 reductase, with a first-order rate constant of 14 min-1 at 25 °s C. During the decay, product was formed (from the substrate methacetin) in 61% theoretical yield, although this reaction requires electron transFe2+r among P450 molecules and may not be related to normal turnover. Steady-state spectra suggest that one or more iron-oxygen complexes accumulate, representing entities between the Fe2+(2+)-O2 complex and putative Fe2+O(3+) entity. Kinetic isotope efFe2+ct experiments were done with several substrates, mainly anisoles. Apparent intrinsic deuterium isotope efFe2+cts as high as 15 were measured. In all cases, the C-H bond-breaking step is at least partially rate-limiting. The isotope efFe2+cts were not strongly attenuated in noncompetitive or competitive experiments, consistent with relatively rapid P450-substrate exchange, except with the active enzyme Fe2+-O complex. Kinetic simulations with the available data (i) are consistent with the view that C-H bond breaking is a major rate-limiting step, (ii) demonstrate that increasing the rate of this step will afFe2+ct kcat, km, and kinetic hydrogen isotope efFe2+cts but will only increase catalytic efficiency to a certain °, (iii) indicate that increasing ground-state binding can increase catalytic efficiency but not kcat, and (iv) suggest that nonproductive binding modes and abortive reduction of O2 are factors that attenuate catalytic efficiency. [ABSTRACT FROM AUTHOR]

The basis for our previous observations [Kaminsky, L. S., Guengerich, F. P., Dannan, G. A. & Aust, S. D. (1983) Arch. Biochem. Biophys. 225, 398–404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4′, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver eytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F > P-450PB-D ≥ P-450UT-A ≥ P-450BNF/ISF-G > P-450PB/PCN-E > P-450PB-B ≥ P-450PB-C ≥ P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations. [ABSTRACT FROM AUTHOR]

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P.450 in animals. The expression of four cytochrome P450 enzymes (or groups of enzymes, namely P450 IIIA. P-450 IICS/9/10, P450 lIE1, and P-450 1A2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses. depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 11C89/10 mRNA transcripts and the corresponding protein, while 3-methytcholanthrene was ineffective. Both P-450 IIlA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIlA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIlA expression was not affected by phenobarbital and was decreased by 3- methyicholanthrene. P-450s 1A2 and IIE1 decreased to 25- 50% of the initial level during these cultures. P-450 1A2 and ethoxyresorufin O-deethylase activity (which is a menooxygenase activity related to P.450 IA family) were increased only by 3-methyleholanthrene and did not respond to the other inducers. P-450 IIEI was not induced by any of these compounds. P-450 IIC8/9/10 and P450 lIlA mRNA levels were also measured in human hepatocytes from one newborn. P450 11C8/9/10 was barely expressed in freshly isolated cells but increased dramaticaily with time in culture. P.450 IIlA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver. [ABSTRACT FROM AUTHOR]

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity, Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16β-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16α increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus. [ABSTRACT FROM AUTHOR]

Metals are present in about one-half of the protein structures available and also have critical roles in nucleic acid biochemistry. This prologue introduces the fourth of the Thematic Minireview Series on Metals in Biology, which deals with several topics involving iron, manganese, copper, and other metals. The six minireviews discuss metal transport and intracellular homeostasis, including chaperones and siderophores, maturation of the diiron active sites in hydrogenases, the balance between manganese and iron, and copper homeostasis relevant to pathogens. [ABSTRACT FROM AUTHOR]

Veith et al. (2009) analyzed over 17,000 compounds for interaction with the five major human cytochrome P450 drug-metabolizing enzymes. Analysis of the results provides some structural insight and framework for expansion. [Copyright &y& Elsevier]

Metals have important roles in biochemistry ranging from essential to toxic. This prologue introduces the second of the Thematic Minireview Series on Metals in Biology, which includes minireviews on five metals: iron, zinc, nickel, vanadium, and arsenic. Three of the minireviews are focused on the roles of the metals in enzymes (iron, nickel, and vanadium). Zinc deficiency is discussed in another, and the arsenic minireview deals with the toxic and some potentially useful applications of the biological effects. [ABSTRACT FROM AUTHOR]

Metals are important in biochemistry, and the concentrations of many are highly regulated. This prologue introduces the thematic series "Metals in Biology," which includes minireviews on three metals: iron, copper, and selenium. Deficiencies and excesses of all three of these metals cause problems in human health. The three minireviews deal with regulation of iron homeostasis, the roles of copper metabolism in cell regulation and disease, and the functions of selenoproteins. [ABSTRACT FROM AUTHOR]

O6-Methyl-2′-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations duringDNAreplication, with different DNApolymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2″-deoxythymidine-5″-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ~4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (kcat and Km) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (kcat/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822–823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations. [ABSTRACT FROM AUTHOR]

DNA polymerase (pol) κ, oneof the Y-family polymerases,has been shown to function in error-free translesion DNA synthesis(TLS) opposite the bulky N2-guanyl DNAlesions induced by many carcinogens such as polycyclic aromatic hydrocarbons.We analyzed the biochemical properties of eight reported human polκ variants positioned in the polymerase core domain, using therecombinant pol κ (residues 1–526) protein and the DNAtemplate containing an N2-CH2(9-anthracenyl)G (N2-AnthG). The truncationR219X was devoid of polymerase activity, and the E419G and Y432S variantsshowed much lower polymerase activity than wild-type pol κ.In steady-state kinetic analyses, E419G and Y432S displayed 20- to34-fold decreases in kcat/Kmfor dCTP insertion opposite G and N2-AnthG compared to that of wild-type pol κ. TheL21F, I39T, and D189G variants, as well as E419G and Y432S, displayed6- to 22-fold decreases in kcat/Kmfor next-base extension from C paired with N2-AnthG, compared to that of wild-type pol κ.The defective Y432S variant had 4- to 5-fold lower DNA-binding affinitythan wild-type, while a slightly more efficient S423R variant possessed2- to 3-fold higher DNA-binding affinity. These results suggest thatR219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variationssubstantially impair the TLS ability of pol κ opposite bulky N2-G lesions in the insertion step opposite thelesion and/or the subsequent extension step, raising the possibilitythat certain nonsynonymous pol κ genetic variations translateinto individual differences in susceptibility to genotoxic carcinogens. [ABSTRACT FROM AUTHOR]

Human polymorphisms in the 5′-upstream regulatory regions and also protein coding regions of cytochrome P450 2E1 (CYP2E1) are known to be associated with several diseases, including cancer and alcohol liver toxicity. In this study, we report novel mutations in the N-terminal protein targeting regions of CYP2E1 that markedly affect subcellular localization of the protein. Variant W23R/W30R protein (termed W23/30R) is preferentially targeted to mitochondria but very poorly to the endoplasmic reticulum, whereas the L32N protein is preferentially targeted to the endoplasmic reticulum and poorly to mitochondria. These results explain the physiological significance of bimodal CYP targeting to the endoplasmic reticulum and mitochondria previously described. COS-7 cells and HepG2 cells stably expressing W23/30R mutations showed markedly increased alcohol toxicity in terms of increased production of reactive oxygen species, respiratory dysfunction, and loss of cytochrome c oxidase subunits and activity. Stable cells expressing the L32N variant, on the other hand, were relatively less responsive to alcohol-induced toxicity and mitochondrial dysfunction. These results further support our previous data, based on mutational studies involving altered targeting, indicating that mitochondria- targeted CYP2E1 plays an important role in alcohol liver toxicity. The results also provide an interesting new link to genetic variations affecting subcellular distribution of CYP2E1 with alcohol-induced toxicity. [ABSTRACT FROM AUTHOR]

Abstract: The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules. [Copyright &y& Elsevier]

1,N²-Etheno(∈)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3′-(1,N²-∈-G)TACT-5′, among the four dNTPs tested individually. With the template 3′-(1,N²-∈-G)CACT-5′, both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3′-(1,N²-∈-G)CACT-5′) the major product was 5′-GTGA-3′ and the minor product was 5′-AGTGA-3′. With the template 3′.(1,N²-∈-G)TACT-5′, the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5′-AATGA-3′, 5′-ATTGA-3′, 5′-ATGA-3′, and 5′-TGA-3′. An x-ray crystal structure of Dpo4 was solved (2.1 Å) with a primer-template and A placed in the primer to be opposite the 1,N²-∈-G in the template 3′-(1,N²-∈-G)TACT 5′. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N²-∈-G in 3′-(1,N²-∈-G)CACT-5′. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N²-∈-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5(b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (kon ∼ 106 M-1 s-1), and the koff rates range from 5.7 to 36 s-1 (at 23 °C). Reduction of ferric P450 2A6 is rapid (7.6 s-1) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k ≥ 106 M-1 s-1), and the putative Fe2+.O2 complex decayed at a rate of ∼0.3 s-1 at 23 °C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by be. Kinetic analyses showed that ∼⅓ of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6–10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. [ABSTRACT FROM AUTHOR]

It has been proposed that the DNA repair protein O6alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive halfmustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s). [ABSTRACT FROM AUTHOR]