The Structural Basis for T-antigen Hydrolysis by Streptococcus pneumoniae

Abstract

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.

Recognition of the Helical Structure of β-1,4-Galactan by a New Family of Carbohydrate-binding Modules

“…The highest structural similarity was found between TmCBM61 and the two mannan-binding family 16 CBMs from Caldanaerobius polysaccharolyticus with root mean square deviations of ϳ1.9 -2.0 Å over the full length of the CBMs (19). A similar root mean square deviation of 1.9 Å (over 141 matched C␣s) was found with residues 1056 -1215 of the Streptococcus pneumoniae endo-␣-N-acetylglucosaminidase (Protein Data Bank code 3ECQ) (29); however, this module has no demonstrated carbohydrate binding function. Significant but more distant structural similarity was found with CBMs from the large number of families that adopt the ␤-sandwich fold.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…The crystal structure of endo--GalNAcase from Streptococcus pneumoniae R6 (EngSP) was determined at 2.9 Å resolution for a selenomethionine-labeled protein. 71) We determined the crystal structure of EngBF at 2.0 Å resolution, and described its structural basis for substrate recognition by mutational and computational analysis. 72,73) Four Mn 2þ ions derived from crystallization solution were found in the crystal structure of EngBF (Fig.…”

Solvents derived from glycerol modify classical regioselectivity in the enzymatic synthesis of disaccharides with Biolacta β-galactosidase

“…This information could explain the synthetic behavior of Biolacta enzymes with high preference 15 to Gal-(1 4)GlcNAc synthesis instead of the classical B. circulans tendency toward (1 3) linkages 37, 38 that does not occur in Biolacta preparation.…”

“…In this case, the acetyl chain is not pushing deeper the acceptor. Apparently, the interaction with the solvent is not as unfavorable as in the case of water only, and the molecule adopts a shallow binding mode with 15 essentially the same interactions of the deep binding mode (Tyr-624 and Lys-664) with the additional interaction with Ser-648. The distances measured during MD simulations are correlated with the experimental results, in which the formation of β(1 6) linkage is expected in 5-water mixture, while the β(1 4) linkage 20 is the main product in water with some β(1 6) linked molecules.…”

“…[15][16][17][18] As the understanding of these biological functions increases, the need for practical 55 synthetic procedures of oligosaccharides in large quantities has become a major subject. Organic chemical methods for obtaining them have been developed, 19-21 but they involve several elaborate protection and deprotection procedures.…”