A "terminal" case of glycan catabolism: Structural and enzymatic characterization of the sialidases of Clostridium perfringens.

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here, we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C.

The structure of a pectin-active family 1 polysaccharide lyase from the marine bacterium Pseudoalteromonas fuliginea.

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1).

A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins.

A Fluorogenic Disaccharide Substrate for α-Mannosidases Enables High-Throughput Screening and Identification of an Inhibitor of the GH92 Virulence Factor from .

Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium has been demonstrated to be an important mechanism of virulence. While employs a multitude of glycan processing enzymes, the -mannosidase SpGH92 has been shown to be an important virulence factor.

Separation and Visualization of Glycans by Fluorophore-Assisted Carbohydrate Electrophoresis.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a method in which a fluorophore is covalently attached to the reducing end of carbohydrates, thereby allowing high-resolution separation by electrophoresis and visualization. This method can be used for carbohydrate profiling and sequencing, as well as for determining the specificity of carbohydrate-active enzymes. Here we describe and demonstrate the use of FACE to separate and visualize the glycans released following digestion of oligosaccharides by glycoside hydrolases (GHs) using two examples: (i) the digestion of chitobiose by the streptococcal β-hexosaminidase GH20C and (ii) the digestion of glycogen by the GH13 member SpuA.