Frédéric Krzewinski

β-1,3-Galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082 : characterization, partial purification and relation to mucin degradation

A new enzyme has been characterized in a cell‐free extract of Bifidobacterium bifidum that catalysed the reversible phosphorolytic cleavage of β‐1,3‐galacto‐oligosaccharides. In the presence of Pi, the phosphorolysis reaction was favoured and was accompanied by a Walden reaction. Cleavage of the β‐glycosidic linkage gave an α‐galactoside derivative (α‐D‐galactose 1‐phosphate). The enzyme possesses a high specificity for β‐D‐galactosido‐(1,3)‐N‐acetylglucosamine and β‐D‐galactosido‐(1,3)‐N‐acetylgalactosamine. This purified intracellular enzyme had an estimated molecular mass of 140 kDa. The galactophosphorolytic activity on disaccharides was optimal at pH 6–6.5 and the reverse reaction was optimal at pH 5.5–6. The temperature optimum for phosphorolysis and the reverse reaction was approx. 50–55 °C. This enzyme is of particular interest in degrading some β‐D‐Gal(1,3) linkages and should be classified as EC 2.4.1.‐.

Characterization of the Lactose Transport System in the Strain Bifidobacterium bifidum DSM 20082

Abstract. Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose 14C. Km and Vmax values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the β-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-β-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na+, Li+, or K+ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the strain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway.

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Glucose and Galactose Transport in Bifidobacterium bifidum DSM 20082

Abstract. Sugar uptake was measured with 3H-galactose and 14C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters KM and Vmax were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K+ ions.

Bifidobacterium longum Requires a Fructokinase (Frk; ATP:d-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N -acetylglucosamine metabolism in Bifidobacterium

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

Characterization of the recombinant Candida albicans β-1,2-mannosyltransferase that initiates the β-mannosylation of cell wall phosphopeptidomannan.

The presence of β-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (β-mannosyltransferase 1), a member of the recently identified β-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic α-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of α-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of α-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal βMan (β-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific β-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.

Mantyl tagged oligo α (1 → 2) mannosides as Candida albicans β-mannosyl transferases substrates: a comparison between synthetic strategies

Fluorescent oligomannosides are important tools for the evaluation of mannosyl transferase activities and selectivities. In a project dealing with Candida albicans β-mannosyl transferases, three mantyl tagged α (1 → 2) oligomannosides were prepared by different ways: using all ester strategy compatible with the presence of an azido group suitable for direct click chemistry; and alternatively using the more classic benzyl protecting groups. Although more elegant, the all ester strategy has shown important limitations: reduced reactivity of mannosyl donors, and 3 → 2 ester migration. Preliminary enzymatic studies have shown that the synthetic oligomannosides are efficient substrate of β-mannosyl transferases.