RESUMEN
The numerous biological roles of LacNAc-based oligosaccharides have led to an increased demand for these structures for biological studies. In this report, an efficient route for the synthesis of beta-galactosides using a bacterial beta-4-galactosyltransferase/-UDP-4'-gal-epimerase fusion protein is described. The lgtB gene from Neisseria meningitidis and the galE gene from Streptococcus thermophilus were fused and cloned into an expression vector pCW. The fusion protein transfers galactose to a variety of different glucose- and glucosamine-containing acceptors, and utilizes either UDP-galactose or UDP-glucose as donor substrates. A crude lysate from Escherichia coli expressing the fusion protein is demonstrated to be sufficient for the efficient preparation of galactosylated oligosaccharides from inexpensive UDP-glucose in a multigram scale. Lysates containing the fusion protein are also found to be useful in the production of more complex oligosaccharides in coupled reaction mixtures, e.g., in the preparation of sialosides from N-acetylglucosamine. Thus, bacterially expressed fusion protein is well suited for the facile and economic preparation of natural oligosaccharides and synthetic derivatives based on the lactosamine core.
Asunto(s)
Galactósidos/biosíntesis , N-Acetil-Lactosamina Sintasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , UDPglucosa 4-Epimerasa/metabolismo , Secuencia de Carbohidratos , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/biosíntesis , N-Acetil-Lactosamina Sintasa/genética , Neisseria meningitidis/enzimología , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Streptococcus/enzimología , UDPglucosa 4-Epimerasa/biosíntesis , UDPglucosa 4-Epimerasa/genética , Uridina Difosfato Glucosa/metabolismoRESUMEN
FlaA1 is a small soluble protein of unknown function in Helicobacter pylori. It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography. Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1. Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.
Asunto(s)
Proteínas Bacterianas , Deshidrogenasas de Carbohidratos/química , Helicobacter pylori/enzimología , Hidroliasas/metabolismo , Oxidorreductasas/metabolismo , Benzaldehídos/farmacología , Western Blotting , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis Capilar , Prueba de Complementación Genética , Glucosa/metabolismo , Histidina/metabolismo , Hidroliasas/química , Hidroliasas/genética , Indicadores y Reactivos/farmacología , Cinética , Espectrometría de Masas , Modelos Químicos , Mutagénesis , Níquel/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Unión Proteica , Espectrofotometría , Especificidad por Sustrato , Factores de TiempoRESUMEN
Proteus mirabilis is a common cause of upper urinary tract infections. Fimbriae-mediated adherence of this organism to urinary tract epithelium and invasion of host cells are factors thought to be important in its pathogenesis. We have assessed the effect of growth in serum, blood, and urine on the ability of P. mirabilis 7570 to adhere to and invade in vitro the cell line EJ/28, derived from a human urinary tract tumour, and to express nonagglutinating fimbriae (NAF). Proteus mirabilis was capable of adhering to EJ/28 cells to varying degrees depending upon the growth conditions used. It was invasive under all conditions, except when grown in urine, and was found to be particularly so when serum or blood was present in the media. Expression of NAF occurred under all growth conditions examined and was limited only by a decrease in temperature.
