Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi

Abstract

The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de-N-acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline b-chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite.

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine

“…In PgaB, this helix (␣2 colored purple in Fig. 1, A and B) occurs internally in the sequence when compared with previously reported structures (21)(22)(23)(24)(25)(26)52). Another difference for PgaB is the presence of four ␤-hairpins, ␤2-3, ␤6 -7, ␤9 -10, and ␤11-12 (Figs.…”

“…In addition to PgaB and CtCE4, two other CE4s, PdaA from Bacillus subtilis and ECU11_510 from Encephalitozoon cuniculi, also contain significant deviations to the canonical CE4 motifs (22,52). PdaA has modifications in MT1 and MT3, whereas ECU11_510 has modification in MT1-MT4 (22,52). This comparison suggests that modifications to CE4 motifs may provide a means to alter substrate specificity, metal preference, or the reaction mechanism.…”

“…The preference of Ni 2ϩ over Zn 2ϩ for PgaB and CtCE4 does not appear to be from altered coordination chemistry, because Zn 2ϩ -bound CE4 structures also have octahedral arrangements. In addition to PgaB and CtCE4, two other CE4s, PdaA from Bacillus subtilis and ECU11_510 from Encephalitozoon cuniculi, also contain significant deviations to the canonical CE4 motifs (22,52). PdaA has modifications in MT1 and MT3, whereas ECU11_510 has modification in MT1-MT4 (22,52).…”

“…CE4s are known for their metal-dependent deacetylation of Oand N-acetylated polysaccharides such as chitin, peptidoglycan, and acetylxylan (20). Characterization of several CE4s has shown that deacetylation is dependent on a variety of divalent metal ions (21)(22)(23)(24)(25)(26), and it is proposed that the deacetylation reaction proceeds through a metal stabilized tetrahedral oxyanion intermediate (23)(24)(25). The exact function of the C-terminal domain of PgaB is unknown; however, it is required for efficient de-N-acetylation of PNAG in vivo (17).…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…CE4 family enzymes are characterised by their dependence, for catalytic activity (deacetylation), on coordination of either a Co 2+ or Zn 2+ cation [68], and by the use of the His-His-Asp metal binding triad [38]. CE4 AcXEs operate an acid (aspartic acid) -base (histidine) catalytic mechanism and are also known as 'NodB homologs' [69][70][71]. They possess the highly conserved catalytic NodB domain [72] characteristic of other rhizobial NodB enzymes in the CE4 family such as the chitin deacetylases, chitooligosaccharide deacetylases, rhizobial nod factor deacetylases as well as peptidoglycan N-acetylglucosamine deacetylases and Nacetylmuramic deacetylases which de-esterify N-or O-acetyl bonds of plant cell wall polymers or oligomers [73][74][75].…”