923 research outputs found
Human Heme Oxygenase Oxidation of 5- and 15-Phenylhemes
Human heme oxygenase-1 (hHO-1) catalyzes the O2- dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the -meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IX . Surprisingly, a 15-methyl substituent caused exclusive cleavage at the -meso- rather than at the normal, unsubstituted -meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IX and oxidizes 15- phenylheme at the -meso position to give 10-phenylbiliverdin IX . The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-Ă
crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141â150 and in the proximal Lys18 and Lys22. In the 5-phenylhemehHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26â42 near the -meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.Fil: Wang, Jingling. University of California; Estados UnidosFil: Niemevz, Fernando. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de QuĂmica OrgĂĄnica; ArgentinaFil: Lad, Latesh. University of California; Estados UnidosFil: Huang, Liusheng. University of California; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de QuĂmica OrgĂĄnica; ArgentinaFil: Buldain, Graciela Yolanda. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂmica. Departamento de QuĂmica OrgĂĄnica; ArgentinaFil: Poulos, Thomas L.. University of California; Estados UnidosFil: Ortiz de Montellano, Paul R.. University of California; Estados Unido
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Xanthates: Metabolism by Flavoprotein-Containing Monooxygenases and Antimycobacterial Activity.
Ethionamide (ETH) plays a central role in the treatment of tuberculosis in patients resistant to the first-line drugs. The ETH, thioamide, and thiourea class of antituberculosis agents are prodrugs that are oxidatively converted to their active S-oxides by the mycobacterial flavin-dependent monooxygenase (EtaA) of Mycobacterium tuberculosis, thus initiating the chain of reactions that result in inhibition of mycolic acid biosynthesis and cell lysis. As part of a search for new lead candidates, we report here that several xanthates are oxidized by purified EtaA to S-oxide metabolites (perxanthates), which are implicated in the antimycobacterial activity of these compounds. This process, which is analogous to that responsible for activation of ETH, is also catalyzed by human flavoprotein monooxygenase 3. EtaA was not inhibited in a time-dependent manner during the reaction. Xanthates with longer alkyl chains were oxidized more efficiently. EtaA oxidized octyl-xanthate (Km = 5 ”M; Vmax = 1.023 nmolP/min; kcat = 5.2 molP/min/molE) more efficiently than ETH (194 ”M; 1.46 nmolP/min; 7.73 nmolP/min/molE, respectively). Furthermore, the in vitro antimycobacterial activity of four xanthates against M. tuberculosis H37Hv was higher (minimum inhibitory concentration of around 1 ”M) than that of ETH (12 ”M)
Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique
AbstractDynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection
Hole Hopping through Tryptophan in Cytochrome P450
Electron-transfer kinetics have been measured in four conjugates of cytochrome P450 with surface-bound Ru-photosensitizers. The conjugates are constructed with enzymes from Bacillus megaterium (CYP102A1) and Sulfolobus acidocaldarius (CYP119). A W96 residue lies in the path between Ru and the heme in CYP102A1, whereas H76 is present at the analogous location in CYP119. Two additional conjugates have been prepared with (CYP102A1)W96H and (CYP119)H76W mutant enzymes. Heme oxidation by photochemically generated Ru^(3+) leads to P450 compound II formation when a tryptophan residue is in the path between Ru and the heme; no heme oxidation is observed when histidine occupies this position. The data indicate that heme oxidation proceeds via two-step tunneling through a tryptophan radical intermediate. In contrast, heme reduction by photochemically generated Ru+ proceeds in a single electron tunneling step with closely similar rate constants for all four conjugates
Quantum mechanics/molecular mechanics modeling of drug metabolism:Mexiletine N-hydroxylation by cytochrome P450 1A2
The mechanism of cytochrome P450Â(CYP)-catalyzed
hydroxylation of
primary amines is currently unclear and is relevant to drug metabolism;
previous small model calculations have suggested two possible mechanisms:
direct N-oxidation and H-abstraction/rebound. We have modeled the
N-hydroxylation of (<i>R</i>)-mexiletine in CYP1A2 with
hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing
a more detailed and realistic model. Multiple reaction barriers have
been calculated at the QMÂ(B3LYP-D)/MMÂ(CHARMM27) level for the direct
N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers
indicate that the direct N-oxidation mechanism is preferred and proceeds
via the doublet spin state of Compound I. Molecular dynamics simulations
indicate that the presence of an ordered water molecule in the active
site assists in the binding of mexiletine in the active site, but
this is not a prerequisite for reaction via either mechanism. Several
active site residues play a role in the binding of mexiletine in the
active site, including Thr124 and Phe226. This work reveals key details
of the N-hydroxylation of mexiletine and further demonstrates that
mechanistic studies using QM/MM methods are useful for understanding
drug metabolism
Calibration of the channel that determines the omega-hydroxylation regiospecificity of cytochrome P4504A1 - Catalytic oxidation of 12-halododecanoic acids
The fatty acid omega-hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in (H2O)-O-18 show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O-2. The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an omega-1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the omega-aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[H-2](2)-12-chlorododecanoic acid causes a 2-3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl >> I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 angstrom but smaller than 4.30 angstrom
Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins
As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O_2 and its derived intermediates such as hydrogen peroxide to afford a variety of metalâoxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metalâoxo species, are the basis for the various biological functions of O_2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O_2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important ironâoxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs
Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.
The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 ÎŒM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.MEK was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. AGC and KJM were supported by grants from the BBSRC (Grant No: BB/I019669/1 and BB/I019227/1). GGJ received funding from the Ogden Trust and the Isaac Newton Trust administered through the University of Cambridge Bursary Scheme. DSCH was supported by a Croucher Cambridge International Scholarship awarded in conjunction between the Croucher Foundation and the Cambridge Overseas Trust. SAH was supported by an Oliphant Cambridge Australia Scholarship (App No: 10132070) awarded by the Cambridge Commonwealth Trust. The contributions of LBM and LPSC were supported by funds from the Francis Crick Institute, which receives its core funding principally from Wellcome Trust, Cancer Research UK, and the UK Medical Research Council (to LPSC - MC_UP_A253_1111) and funds from FAPESP, CNPq and CAPES-PDSE (to LBM - 2011/21232-1, 140079/2013-0, 99999.003125/2014-09).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acs.jmedchem.6b0000
Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study
The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme
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