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1.
Environ Microbiol ; 19(12): 4913-4925, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28925551

RESUMEN

Microbial reduction of nitrous oxide (N2 O) is an environmentally significant process in the biogeochemical nitrogen cycle. However, it has been recognized only recently that the gene encoding N2 O reductase (nosZ) is organized in varying genetic contexts, thereby defining clade I (or 'typical') and clade II (or 'atypical') N2 O reductases and nos gene clusters. This study addresses the enzymology of the clade II Nos system from Wolinella succinogenes, a nitrate-ammonifying and N2 O-respiring Epsilonproteobacterium that contains a cytochrome c N2 O reductase (cNosZ). The characterization of single non-polar nos gene deletion mutants demonstrated that the NosG, -C1, -C2, -H and -B proteins were essential for N2 O respiration. Moreover, cells of a W. succinogenes mutant lacking a putative menaquinol-oxidizing Rieske/cytochrome bc complex (QcrABC) were found to be incapable of N2 O (and also nitrate) respiration. Proton motive menaquinol oxidation by N2 O is suggested, supported by the finding that the molar yield for W. succinogenes cells grown by N2 O respiration using formate as electron donor exceeded that of fumarate respiration by about 30%. The results demand revision of the electron transport chain model of clade II N2 O respiration and challenge the assumption that NosGH(NapGH)-type iron-sulfur proteins are menaquinol-reactive.


Asunto(s)
Citocromos b/genética , Citocromos c/genética , Complejo III de Transporte de Electrones/genética , Transporte de Electrón/genética , Óxido Nitroso/metabolismo , Oxidorreductasas/genética , Wolinella/metabolismo , Citoplasma/metabolismo , Transporte de Electrón/fisiología , Fumaratos/metabolismo , Familia de Multigenes/genética , Nitratos/metabolismo , Oxidación-Reducción , Wolinella/enzimología , Wolinella/genética
2.
Angew Chem Int Ed Engl ; 56(19): 5358-5362, 2017 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-28378514

RESUMEN

Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.


Asunto(s)
Temperatura , Transferasas/química , Transcetolasa/química , Biocatálisis , Estabilidad de Enzimas , Geobacillus stearothermophilus/enzimología , Cetoácidos/química , Cetoácidos/metabolismo , Modelos Moleculares , Estructura Molecular , Mutación , Transferasas/genética , Transferasas/metabolismo , Transcetolasa/genética , Transcetolasa/metabolismo
3.
PLoS One ; 11(2): e0149145, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26886195

RESUMEN

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained Km and kcat values of the recombinant enzyme were 94.3±1.8 µM and 0.39±0.03 s(-1), respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (Ki of 0.1 mM and 0.18 mM). Negligible influence of metals on ß-lactamase activity ruled out that Sml-1 is a Zn2+-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.


Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Streptomyces/enzimología , Transglutaminasas/metabolismo , beta-Lactamasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biotinilación , Dominio Catalítico , Escherichia coli/metabolismo , Espacio Extracelular/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/biosíntesis , Especificidad por Sustrato , beta-Lactamasas/química
5.
AIDS Educ Prev ; 20(5): 435-53, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18956984

RESUMEN

This study quantitatively and qualitatively described HIV risk behaviors among Vietnamese female sex workers (FSWs) who work at three distinct venues in Ho Chi Minh City: street, massage parlors, and bars/clubs. Although 35% of the participants had never been tested for HIV, 18% of street and 7% of bar/club FSWs reported being positive. Almost all massage parlor FSWs had never used a condom for oral sex. Inconsistent condom use for vaginal sex with customers was more prevalent among bar/club FSWs (85%) than massage parlor (72%) and street FSWs (68%). Many participants reported difficulties in negotiating condom use with customers because of economic pressure, maintaining relationships, and lack of bargaining power. Bar/club FSWs revealed a difficult situation where drinking is part of their work. Thirty percent of street FSWs had injected drugs and reported addiction to heroin in relation to their helpless condition as FSWs. Street FSWs had the lowest levels of self-esteem and norms toward practicing safe sex and the highest levels of economic pressure. This study recommends future HIV prevention programs for FSWs in Vietnam that target their specific risk behaviors and work environments.


Asunto(s)
Infecciones por VIH/prevención & control , Conocimientos, Actitudes y Práctica en Salud , Trabajo Sexual , Sexo Inseguro/estadística & datos numéricos , Adolescente , Adulto , Condones/estadística & datos numéricos , Estudios Transversales , Femenino , Humanos , Entrevistas como Asunto , Trastornos Relacionados con Sustancias/epidemiología , Vietnam/epidemiología
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