Structural and enzymatic characterization of the sialidase SiaPG from Porphyromonas gingivalis

Abstract

The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Å resolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.

Functional and structural analyses reveal that a dual domain sialidase protects bacteria from complement killing through desialylation of complement factors

“…The small N-terminal domain (residues 31 through 176) is a putative carbohydrate binding module (CBM), while the C-terminal domain (residues 177-523) exhibits a canonical six-bladed β-propeller sialidase fold, with each blade composed of 3-4 antiparallel β-strands. The overall structure of unliganded PG0352 is very similar to a recent report (41).…”

“…the crystallization cocktail (S4, S5E and S6 Figs). Similarly, a recent report also found a tartrate molecule at the same position [41]. One of the carboxyethyl groups of citrate interacts with Arg-194, Arg-398, and Arg-460, a triad of conserved arginine residues implicated in stabilizing the carboxylate group of sialic acid [48,49,57].…”

“…The active site cleft is located at the center of the β-propeller and is surface accessible ( Fig 5 ). As observed previously, there is a difference in the electrostatic potential on each face of the enzyme, with a more positive potential on the catalytic face and a more negative potential on the opposite face [ 41 ]. This charge differential is proposed to be crucial in orienting the catalytic face of the enzyme towards the substrate [ 51 – 53 ].…”

“…This charge differential is proposed to be crucial in orienting the catalytic face of the enzyme towards the substrate [ 51 – 53 ]. The positions of the side chains of Tyr-488 and Glu-382 suggest that they serve as the nucleophile and the general base catalyst, respectively, as they occupy topologically equivalent positions in other structurally characterized bacterial sialidase enzymes ( Fig 6 ) [ 34 , 41 , 44 , 49 , 54 ]. Similarly, Asp-219 is positioned to function as the acid/base catalyst to activate a water molecule for release of free sialic acid [ 48 , 49 , 55 , 56 ].…”

“…During the preparation of this manuscript, the structure of PG0352 bound to tartrate was published (41). The structure was determined to 2.10Å in space group P3 1 2 1 , with 3 molecules in the asymmetric unit.…”