Stable carboxylic acid derivatized alkoxy silanes.

A convenient and straightforward one-pot hydrosilylation reaction of different unsaturated carboxylic acids with trialkoxysilanes in the presence of catalytic amounts of platinum(IV) dioxide resulted in excellent yields in organofunctional silanes combining carboxy- and alkoxy groups within one molecule.

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis.

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.

Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose.

The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain d-glycero-d-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-d-glycero-d-manno-heptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of d-sedoheptulose 7-phosphate to d-glycero-d-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form d-glycero-d-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate d-glycero-alpha-d-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-d-glycero-alpha-d-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-d-glycero-alpha-d-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of l-glycero-d-manno-heptose.

Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T.

The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria. The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli. The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed. GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor. The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor. The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose. This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase. In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities. So far, no enzyme catalyzing these two types of reactions has been identified. Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family.

Structural and functional analyses of the secondary cell wall polymer of Bacillus sphaericus CCM 2177 that serves as an S-layer-specific anchor.

Sacculi of Bacillus sphaericus CCM 2177 contain a secondary cell wall polymer which was completely extracted with 48% hydrofluoric acid. Nuclear magnetic resonance analysis showed that the polymer is composed of repeating units, as follows: -->3)-[4, 6-O-(1-carboxyethylidene)]( approximately 0. 5)-beta-D-ManpNAc-(1-->4)-beta-D-GlcpNAc-(1-->. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.