Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC.

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Paenibacillus barcinonensis xylanase 10C containing the CBM22-1-CBM22-2 tandem.

A construct containing the CBM22-1-CBM22-2 tandem forming the N-terminal domain of Paenibacillus barcinonensis xylanase 10C (Xyn10C) has been purified and crystallized. A xylan-binding function and an affinity for mixed ß-1,3/ß-1,4 glucans have previously been demonstrated for some members of the CBM22 family. The sequence of the tandem is homologous to the N-terminal domains found in several thermophilic enzymes. Crystals of this tandem were grown by the streak-seeding method after a long optimization strategy. The structure has been determined by molecular replacement to a resolution of 2.43 Å and refinement is under way. This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine-tuning substrate affinity.

Crystallization and preliminary X-ray diffraction analysis of Xyn30D from Paenibacillus barcinonensis.

Xyn30D, a new member of a recently identified group of xylanases, has been purified and crystallized. Xyn30D is a bimodular enzyme composed of an N-terminal catalytic domain belonging to glycoside hydrolase family 30 (GH30) and a C-terminal family 35 carbohydrate-binding domain (CBM35) able to bind xylans and glucuronic acid. Xyn30D shares the characteristic endo mode of action described for GH30 xylanases, with the hydrolysis of the ß-(1,4) bonds of xylan being directed by α-1,2-linked glucuronate moieties, which have to be placed at the -2 subsite of the xylanase active site. Crystals of the complete enzyme were obtained and a full data set to 2.3 Šresolution was collected using a synchrotron X-ray source. This represents the first bimodular enzyme with the domain architecture GH30-CBM35. This study will contribute to the understanding of the role that the different xylanases play in the depolymerization of glucuronoxylan.