Polysaccharide binding sites in hyaluronate lyase – crystal structures of native phage–encoded hyaluronate lyase and its complexes with ascorbic acid and lactose

Abstract

Hyaluronate lyases are a class of endoglycosaminidase enzymes with a high level of complexity and heterogeneity. The main function of the Streptococcus pyogenes bacteriophage protein hyaluronate lyase, HylP2, is to degrade hyaluronan into unsaturated disaccharide units. HylP2 was cloned, over‐expressed and purified to homogeneity. The recombinant HylP2 exists as a homotrimer with a molecular mass of approximately 110 kDa under physiological conditions. The HylP2 was crystallized and the crystals were soaked in two separate reservoir solutions containing ascorbic acid and lactose, respectively. The crystal structures of native HylP2 and its two complexes with ascorbic acid and lactose have been determined. HylP2 folds into four distinct domains with a central core consisting of 16 antiparallel β‐strands forming an irregular triangular tube designated as triple‐stranded β‐helix. The structures of complexes show that three molecules each of ascorbic acid and lactose bind to protein at the sugar binding groove in the triple‐stranded β‐helix domain. Both ascorbic acid and lactose molecules occupy almost identical subsites in the long saccharide binding groove. Both ligands are involved in several hydrogen bonded interactions at each subsite. The binding characteristics and stereochemical properties indicate that Tyr264 may be involved in the catalytic activity of HylP2. The mutation of Tyr264 to Phe264 supports this observation.

Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers

“…Comparable structures include the triple β-helix regions of phage T4 gp12 (41) and gp5 (42), the K1F endosialidase (43) and endo-N-acetylneuramidase (29), streptococcal (pro)phage HylP1 and Hylp2 (44,45), the phage P22 cell penetrating needle gp26 (40), and phage Phi29 tail-spike (46). However, the most similar structures are those of phage P2 gpV (Fig.…”

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

“…The crystal structures of PLs in 18 families have been determined, and the structure and functional relationships of enzymes such as pectin lyase in family PL-1 (14); pectate lyases in families PL-2, PL-3, and PL-10 (15-17); alginate lyases in families PL-5, PL-7, and PL-14 (13,18,19); rhamnogalacturonan lyases in families PL-4 and PL-11 (20,21); chondroitin lyases in families PL-6 and PL-8 (22); hyaluronan lyases in families PL-8 and PL-16 (23,24); heparin lyases in families PL-13 and PL-21 (25,26); and xanthan lyase in family PL-8 (12) have been demonstrated. On the other hand, no structural information on family PL-15 alginate lyases has been accumulated, and the enzyme mechanism for releasing unsaturated monosaccharides from the polysaccharide main chain remains to be clarified.…”

Bacteriophages and Phage-Derived Proteins – Application Approaches

“…Contrarily, the streptococcal proteins belong to a different polysaccharide lyase group, the PL8 family. To date, at least several phage HyaLs have been identified which demonstrate activity against streptococci including hylP from phages H4489A [176, 179], HylP1 (phage 370.1) [174], HylP2 (phage 10403) [177, 180], HylP3 [181] and SEQ2045 [182]. They are mainly found in phages invading two species of group A streptococci: S. pyogenes and S. equi .…”