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 µM; Vmax = 1.023 nmolP/min; kcat = 5.2 molP/min/molE) more efficiently than ETH (194 µM; 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 µM) than that of ETH (12 µM)

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-Å 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 141–150 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 26–42 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 Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Lad, Latesh. University of California; Estados UnidosFil: Huang, Liusheng. University of California; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Buldain, Graciela Yolanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Poulos, Thomas L.. University of California; Estados UnidosFil: Ortiz de Montellano, Paul R.. University of California; Estados Unido

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

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway

Cytochrome P450-activated prodrugs

A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues