RESUMEN
Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.
Asunto(s)
Aeromonas hydrophila/enzimología , Proteínas Bacterianas , Cefamicinas/metabolismo , Cefamicinas/farmacología , Moxalactam/metabolismo , Moxalactam/farmacología , beta-Lactamasas/metabolismo , Cefamicinas/química , Hidrólisis , Cinética , Estructura Molecular , Moxalactam/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Inhibidores de beta-LactamasasRESUMEN
The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.
Asunto(s)
Bacillus/enzimología , Nucleótidos/metabolismo , Oligosacáridos/síntesis química , Azúcares de Uridina Difosfato/química , beta-Galactosidasa/metabolismo , Bioquímica/métodos , Secuencia de Carbohidratos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Nucleótidos/química , Oligosacáridos/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Temperatura , Azúcares de Uridina Difosfato/síntesis química , Azúcares de Uridina Difosfato/metabolismo , beta-Galactosidasa/químicaRESUMEN
The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.
Asunto(s)
N-Acetil-Lactosamina Sintasa/química , Uridina Difosfato N-Acetilglucosamina/química , Azúcares de Uridina Difosfato/síntesis química , Fosfatasa Alcalina/química , Animales , Secuencia de Carbohidratos , Bovinos , Humanos , Lactalbúmina/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Leche/química , Leche Humana/química , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
The trisaccharide Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was enzymatically synthesized, with in situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4'-epimerase, UDP-Gal:GlcNAc beta 4-galactosyltransferase and UDP-Gal:Gal beta 1-->4GlcNAc alpha 3-galactosyltransferase, Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was formed in a 2.2 mumol ml-1 yield starting from the acceptor GlcNAc beta 1-->O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc epitope which pays an important role in various biological and immunological processes.
