Activation by Allostery in Cell-Wall Remodeling by a Modular Membrane-Bound Lytic Transglycosylase from Pseudomonas aeruginosa

ABSTRACTThe peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltG’s ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

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Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of Pseudomonas aeruginosa

ACS Chemical Biology ◽

10.1021/acschembio.6b00194 ◽

2016 ◽

Vol 11 (6) ◽

pp. 1525-1531 ◽

Cited By ~ 14

Author(s):

Mijoon Lee ◽

Teresa Domínguez-Gil ◽

Dusan Hesek ◽

Kiran V. Mahasenan ◽

Elena Lastochkin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Lytic Transglycosylase

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Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of Pseudomonas aeruginosa

ACS Chemical Biology ◽

10.1021/acschembio.6b00756 ◽

2016 ◽

Vol 11 (10) ◽

pp. 2936-2936 ◽

Cited By ~ 3

Author(s):

Mijoon Lee ◽

Teresa Domínguez-Gil ◽

Dusan Hesek ◽

Kiran V. Mahasenan ◽

Elena Lastochkin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Lytic Transglycosylase

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Cell-Wall Remodeling by the Zinc-Protease AmpDh3 from Pseudomonas aeruginosa

Journal of the American Chemical Society ◽

10.1021/ja407445x ◽

2013 ◽

Vol 135 (34) ◽

pp. 12604-12607 ◽

Cited By ~ 32

Author(s):

Mijoon Lee ◽

Cecilia Artola-Recolons ◽

César Carrasco-López ◽

Siseth Martínez-Caballero ◽

Dusan Hesek ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Cell Wall Remodeling ◽

Zinc Protease

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Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103740118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2103740118

Author(s):

Atsushi Taguchi ◽

Julia E. Page ◽

Ho-Ching Tiffany Tsui ◽

Malcolm E. Winkler ◽

Suzanne Walker

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Single Amino Acid ◽

Lytic Transglycosylases ◽

Lipid Ii ◽

Lytic Transglycosylase ◽

Peptidoglycan Cell Wall ◽

Muramidase Activity ◽

Membrane Bound

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

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Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801298115 ◽

2018 ◽

Vol 115 (17) ◽

pp. 4393-4398 ◽

Cited By ~ 11

Author(s):

Mijoon Lee ◽

María T. Batuecas ◽

Shusuke Tomoshige ◽

Teresa Domínguez-Gil ◽

Kiran V. Mahasenan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Catalytic Domain ◽

Bacterial Pathogen ◽

Major Constituent ◽

Wild Type ◽

X Ray ◽

Lytic Transglycosylase ◽

Glycan Chain ◽

Bond Scission

β-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non–cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

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