Muropeptide recycling in Bacillus subtilis : beta-N-acetylglucosaminidase NagZ operates by a unique Asp-His catalytic dyad mechanism

Abstract

The murein (peptidoglycan) is a heteropolymer made up of glycan strands that are cross-linked by short peptides. We identified a pathway in B. subtilis which recovers the N-acetylglucosamine-N-acetylmuramic acid-peptides (muropeptides) derived from the peptidoglycan during growth. This pathway involves orthologs of enzymes involved in MurNAc dissimilation and murein recycling in E. coli: the MurNAc-6-phosphate etherase (MurQ), the MurNAc-6-phosphate-specific transcriptional repressor (MurR) and the MurNAc-specific phosphotransferase system (MurP). In this thesis two further enzymes of this pathway were investigated whose genes are organized in a putative operon along with genes encoding the above mentioned enzymes. The first gene, nagZ (formerly ybbD), was shown to encode a beta-N-acetylglucosaminidase that is identical to an enzyme which was described in the early 1970s, but the corresponding gene was not identified at that time. We showed that the enzyme hydrolyzes the terminal non-reducing N-acetylglucosamine (GlcNAc) of muropeptides as analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and reversed-phase high performance liquid chromatography (RP-HPLC). It also hydrolyzes the glycosidic bond of the chromogenic substrate 4-nitrophenyl-beta-N-acetyl-beta-D-glucosaminide (pNP-beta-GlcNAc) and the fluorogenic substrate 4-methylumbelliferyl-beta-N-acetyl-D-glucosaminide (4-Mu-beta-GlcNAc). KM values of 172 and 110 µM and kcat values of 9.9 and 6.4 s-1 for pNP-beta-GlcNAc and 4-Mu-beta-GlcNAc, respectively, were determined. The optimum pH ranges between pH 5.8 and 6.2. NagZ is secreted and non-covalently bound to the cell wall and can be released from particulate material by high concentrations of salt. NagZ is expressed in the late exponential growth phase and reaches 6-fold higher activity in the stationary or autolysis phase and beside that it is the major exo-beta-N-acetylglucosaminidase of B. subtilis. The second enzyme, AmiE, is also secreted and was identified as a novel amidase that hydrolyzes the N-acetylmuramyl-L-alanine bond of MurNAc-peptides but not of muropeptides. Therefore the enzyme requires the activity of NagZ and together the enzymes liberate MurNAc from muropeptides by sequential hydrolysis. The beta-N-acetylglucosaminidase NagZ of B. subtilis belongs to family 3 of glycoside hydrolases. Family 3 of glycoside hydrolases primarily consists of beta-glucosidases and exo-glucanases which catalyze the hydrolysis of the glycosidic linkages of their substrates via a two-step double-displacement mechanism under retention of their anomeric configuration. In general retaining glycosidases harbour two carboxyl groups in the active site functioning as nucleophile and general acid/base catalyst. The catalytic nucleophile, an aspartate, is conserved within all family 3 glycosidases, whereas the general acid/base catalyst, a glutamate, was only identified for the beta-glucosidases, but is absent in the beta-N-acetylglucosaminidases. The beta-N-acetylglucosaminidases generate a sub-group within this family and are characterized by the conserved D-(ST)-H motif. In this thesis the first three-dimensional structure of a two domain beta-N-acetylgluco-saminidase of family 3 glycosidases, NagZ, was determined with and without the transition state inhibitor O-(2-Acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) bound to the active site with a resolution of 1.7 and 1.4 Å, respectively. The structure revealed an N-terminal (alpha/beta)8-TIM-barrel and a C-terminal alpha-beta-alpha-sandwich domain. From the structure and kinetic analyses an Asp-His dyad was shown to be involved in hydrolysis of O-glycosidic linkages. Mutational studies further confirmed that His234 of the dyad functions as general acid/base catalyst and is coordinated by Asp232. Replacement of the residues His234 or Asp232 with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4-Mu-beta-GlcNAc and affected the pH activity profile consistent with the role as acid/base catalyst. Furthermore the glycosyl-enzyme intermediate accumulated in the His234Gly mutant. The Asp-His dyad is conserved in all beta-N-acetylglucosaminidases, whereas in beta-glucosidases a conserved glutamate is located at the position of the dyad and protrudes from the second domain into the active site. The function of the C-terminal alpha-beta-alpha-sandwich domain in NagZ could not yet be identified. NagZ of B. subtilis is the first enzyme in which an Asp-His dyad is involved in hydrolysis of O-glycosidic bonds resembling the catalytic Asp-His-Ser triad of serine proteases.