Structural Determinants in Streptococcal Unsaturated Glucuronyl Hydrolase for Recognition of Glycosaminoglycan Sulfate Groups

Abstract

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substratebound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.

Crystal Structure of a Bacterial Unsaturated Glucuronyl Hydrolase with Specificity for Heparin

“…Specific inhibitors of UGL are therefore expected to provide anti-bacterial drugs with no side effects. In addition, we demonstrated inducible mRNA expression of the streptococcal enzyme in the presence of GAG (16) and identified structural determinants of the preference of streptococcal UGL for sulfated substrates with 1,3-glycosidic bonds (12). However, the enzyme recognition mechanism for 1,4-glycosidic bond-type substrates from heparin and heparan sulfate remains unknown.…”

“…1B) (11). Carbon atoms C-3, C-4, C-5, and C-6 of unsaturated GlcUA (⌬GlcUA) and IdoUA (⌬IdoUA) are located in a single plane because of the formation of double bonds between C-4 and C-5 (12). Because GlcUA is a C-5 epimer of IdoUA, ⌬GlcUA and ⌬IdoUA are identical in structure.…”

“…Purification-Purification of Streptococcus agalactiae UGL (SagUGL), S. pneumoniae UGL (SpnUGL), Streptococcus pyogenes UGL (SpyUGL), SagUGL mutant S368G (12), and BacillusUGL were expressed and purified as described previously (12,16,21). E. coli cells harboring pET21b-Phep_2238, pET21b-Phep_2649, or pCold IV-Phep_2830 plasmid were collected by centrifugation at 6700 ϫ g for 10 min at 4°C.…”

Metabolic Fate of Unsaturated Glucuronic/Iduronic Acids from Glycosaminoglycans

“…More recently, streptococcal UGL has been demonstrated to act on various unsaturated disaccharides, such as hyaluronan, chondroitin sulfate, heparin, and heparan sulfate, and to release unsaturated uronic acids and amino sugars (9,14). Due to the lack of the hydroxyl group at C4, unsaturated glucuronic (⌬GlcUA) and iduronic acids (⌬IdoUA) are chemically identical (Fig.…”

“…We have identified unsaturated glucuronyl hydrolase (UGL), which is essential for degrading unsaturated disaccharides to constituent monosaccharides (i.e. unsaturated uronic acids and amino sugars) in bacteria, including streptococci (8,9), and found that a genome segment termed the UGL genetic cluster is responsible for the depolymerization, import, and degradation of glycosaminoglycans in streptococci, such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes (10) (see Fig. 2A).…”