Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.

The enzyme-catalysed reactions involved in formation of the bicyclic clavam and carbapenem nuclei, including beta-amino acid and beta-lactam formation, are discussed and compared with those involved in penicillin and cephalosporin biosynthesis. The common role of unusual oxidation reactions in the biosynthetic pathways and the lack of synthetic reagents available to effect them are highlighted.

endo-Glycoceramidase, a membrane-associated family 5 glycosidase, deviates from the typical polysaccharide substrate specificity of other soluble members of the family, preferentially hydrolyzing glycosidic linkages between the oligosaccharide and ceramide moieties of gangliosides. Here we report the first x-ray crystal structures of an endo-glycoceramidase from Rhodococcus sp., in the apo form, in complex with the ganglioside G M3 (Svennerholm ganglioside nomenclature (Svennerholm, L. (1964) J. Lipid Res. 5, 145-155)), and trapped as a glycosyl-enzyme intermediate. These snapshots provide the first molecular insight into enzyme recognition and association with gangliosides, revealing the structural adaptations necessary for glycosidase-catalyzed hydrolysis and detailing a novel ganglioside binding topology. Consistent with the chemical duality of the substrate, the active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains. The specific interactions with the ceramide polar head group manifest a surprising aglycone specificity, an observation substantiated by our kinetic analyses. Collectively, the reported structural and kinetic data provide insight toward rational redesign of the synthetic glycosynthase mutant of endo-glycoceramidase to enable facile synthesis of nonnatural, therapeutically useful gangliosides.

Streptococcus pneumoniae endo-␣-N-acetylgalactosaminidase is a cell surface-anchored glycoside hydrolase from family GH101 involved in the breakdown of mucin type O-linked glycans. The 189-kDa mature enzyme specifically hydrolyzes the T-antigen disaccharide from extracellular host glycoproteins and is representative of a broadly important class of virulence factors that have remained structurally uncharacterized due to their large size and highly modular nature. Here we report a 2.9 Å resolution crystal structure that remarkably captures the multidomain architecture and characterizes a catalytic center unexpectedly resembling that of ␣-amylases. Our analysis presents a complete model of glycoprotein recognition and provides a basis for the structure-based design of novel Streptococcus vaccines and therapeutics.

The initial step in the biosynthesis of the clinically important ␤-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N 2 -(2-carboxyethyl)arginine, a ␤-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP 2 )-dependent enzyme N 2 -(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N 2 -(2-carboxyethyl)arginine synthase, complexed with ThP 2 and Mg 2؉ , to 2.35-Å resolution. The structure was solved in two space groups, P2 1 2 1 2 1 and P2 1 2 1 2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP 2 and Mg 2؉ cofactors are present at the active site, with ThP 2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the D-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP 2 . The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative ␤-amino acids.