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

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Litzinger, Silke
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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.

Zusammenfassung in einer weiteren Sprache

Das Murein (Peptidoglykan) ist ein Heteropolymer bestehend aus Glykansträngen, die über kurze Peptidketten quervernetzt sind. Wir haben einen Abbauweg in Bacillus subtilis identifiziert, über den N-Acetylglukosamin-N-Acetylmuramisäure-Peptide (Muropeptide), freigesetzt aus dem Peptidoglykan während des Wachstums, regeneriert werden. Dieser Abbauweg beinhaltet Orthologe zu Enzymen des Stoffwechsels von MurNAc sowie des Recyclings von Murein in E. coli: die MurNAc-6-Phosphat Etherase (MurQ), den MurNAc-6-Phosphat-spezifischen transkriptionellen Repressor (MurR) und das MurNAc-spezifische Phosphotransferase- System (MurP). In dieser Arbeit wurden zwei weitere Enzyme untersucht, deren Gene mit den Genen der oben genannten Enzyme in einem möglichen Operon organisiert sind. Es wurde gezeigt, dass das erste Gen, nagZ (früher ybbD), für eine beta-N-Acetylglukosaminidase kodiert, die in den frühen 70er Jahren beschrieben wurde, jedoch konnte ihr zu jenem Zeitpunkt noch kein Gen zugeordnet werden. Wir haben anhand von fluorophore-assisted carbohydrate electrophoresis (FACE) und reversed-phase high performance liquid chromatography (RP-HPLC) gezeigt, dass NagZ das endständige nicht-reduzierende N-Acetylglukosamin (GlcNAc) aus Muropeptiden freisetzt. Weiterhin hydrolysiert es die glykosidische Bindung des chromogenen Substrates 4-nitrophenyl-beta-N-acetyl-D-glucosaminide (pNP-beta-GlcNAc) sowie des fluorogenen Substrates 4-methylumbelliferyl-beta-N-acetyl-D-glucosaminide (4-Mu-beta-GlcNAc). KM Werte von jeweils 172 und 110 µM sowie kcat Werte von jeweils 9.9 und 6,4 s-1 für pNP-beta-GlcNAc und 4-Mu-beta-GlcNAc wurden bestimmt. Das pH-Optimum liegt in einem Bereich von 5,8 bis 6,2. NagZ wird sekretiert, ist nicht-kovalent an die Zellwand gebunden und kann von partikulärem Material durch hohe Salzkonzentrationen freigesetzt werden. NagZ wird in der späten exponentiellen Wachstumsphase exprimiert und erreicht eine 6-fach höhere Aktivität in der stationären bzw. Autolyse-Phase und ist zudem die wichtigste exo-beta-N-Acetylglukosaminidase von B. subtilis. Das zweite Enzym, AmiE, wird ebenfalls sekretiert und wurde als neue Amidase identifiziert, die die N-Acetylmuramyl-L-Alanin Bindung von MurNAc-Peptiden spaltet, nicht aber von Muropeptiden. Daher benötigt das Enzym die Aktivität von NagZ, so dass MurNAc von Muropeptiden durch sequentielle Hydrolyse beider Enzyme gemeinsam freigesetzt wird. Die beta-N-Acetylglukosaminidase NagZ aus B. subtilis gehört zur Familie 3 der Glykosid-Hydrolasen. Die Familie 3 der Glykosid-Hydrolasen besteht hauptsächlich aus beta-Glukosidasen und exo-Glukanasen, die die Hydrolyse der glykosidischen Bindung ihrer Substrate unter Retention ihrer anomerischen Konfiguration durch einen Zwei-Stufen-Mechanismus katalysieren. Im Allgemeinen besitzen Glykosidasen, die unter Retention arbeiten, im aktiven Zentrum je zwei Carboxyl-Gruppen, die als Nukleophil und Säure/Base-Katalysator fungieren. Das katalytische Nukleophil, ein Aspartat, ist innerhalb aller Familie-3-Glykosidasen konserviert, während der allgemeine Säure/Base-Katalysator, ein Glutamat, nur für die beta-Glukosidasen identifiziert wurde, nicht aber in den beta-N-Acetylglukosaminidasen. Die beta-N-Acetylglukosaminidasen bilden eine Untergruppe innerhalb dieser Familie, die durch das konservierte D-(ST)-H-Motiv charakterisiert ist. In dieser Arbeit wurde die erste Struktur einer beta-N-Acetylglukosaminidase mit zwei Domänen der Familie-3-Glykosidasen, NagZ, gelöst. Die Struktur wurde sowohl mit als auch ohne gebundenen Übergangszustand-Inhibitor PUGNAc (O-(2-Acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate) im aktiven Zentrum mit einer Auflösung von jeweils 1,7 und 1,4 Å gelöst. Die Struktur zeigte eine N-terminale (alpha/beta)8-TIM-Barrel- und eine C-terminale alpha-beta-alpha-Sandwich-Domäne. Die Struktur und kinetische Analysen zeigten, dass eine Asp-His-Diade in der Hydrolyse O-glykosidischer Bindungen involviert ist. Mutationen dieser Reste bestätigten, dass His234 der Diade als Säure/Base-Katalysator fungiert, der durch Asp232 koordiniert wird. Der Austausch des Restes His234 oder Asp232 durch Glycin reduzierte die Geschwindigkeit der Hydrolyse des fluorogenen Substrates 4-Mu-beta;-GlcNAc und beeinträchtigte das pH-Aktivitätsprofil übereinstimmend mit der Rolle als Säure/Base-Katalysator. Darüberhinaus akkumulierte das Glykosyl-Enzym-Zwischenprodukt in der His234Gly Mutante. Die Asp-His-Diade ist in allen beta-N-Acetylglukosaminidasen konserviert, während in den beta-Glukosidasen anstelle der Diade ein konservierter Glutamat-Rest lokalisiert ist, der von der zweiten Domäne in das aktive Zentrum ragt. Die Funktion der C-terminalen alpha-beta-alpha-Sandwich Domäne in NagZ konnte nicht geklärt werden. NagZ von B. subtilis ist das erste Enzym, bei dem eine Asp-His-Diade an der Hydrolyse O-glykosidischer Bindungen beteiligt ist und zudem der katalytischen Asp-His-Ser-Triade von Serinproteasen ähnelt.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Bacillus subtilis, Zellwand-Recycling, Kristallstruktur BsNagZ, Mechanismus BsNagZ, Bacillus subtilis, cell wall recycling, crystal structure BsNagZ, mechanism BsNagZ
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ISO 690LITZINGER, Silke, 2009. Muropeptide recycling in Bacillus subtilis : beta-N-acetylglucosaminidase NagZ operates by a unique Asp-His catalytic dyad mechanism [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Litzinger2009Murop-7888,
  year={2009},
  title={Muropeptide recycling in Bacillus subtilis : beta-N-acetylglucosaminidase NagZ operates by a unique Asp-His catalytic dyad mechanism},
  author={Litzinger, Silke},
  address={Konstanz},
  school={Universität Konstanz}
}
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