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J Biol Chem. 2018 Apr 20;293(16):6000-6010. doi: 10.1074/jbc.RA117.001095. Epub 2018 Feb 26.

A step-by-step in crystallo guide to bond cleavage and 1,6-anhydro-sugar product synthesis by a peptidoglycan-degrading lytic transglycosylase.

Author information

1
From the Institut Pasteur, Département de Microbiologie, Unité Biologie et Génétique de la Paroi Bactérienne, 75015 Paris, France, awilliam@pasteur.fr.
2
INSERM, 75015 Paris, France.
3
From the Institut Pasteur, Département de Microbiologie, Unité Biologie et Génétique de la Paroi Bactérienne, 75015 Paris, France.
4
the Gustave Roussy Comprehensive Cancer Center, Tumour Immunology and Immunotherapy, F-94805 Villejuif, France.
5
the Institut Pasteur, CNRS USR 2000, Unité des Spectrométrie de Masse Structurale et Proteomique, 75015 Paris, France.
6
the Institut Pasteur, Plate-forme de Cristallographie, CNRS-UMR3528, 75724 Paris, France, and.
7
the Institut Pasteur, Département d'Infection et Epidémiologie, Unité des Infection Bactériennes Invasives, 75015 Paris, France.
8
From the Institut Pasteur, Département de Microbiologie, Unité Biologie et Génétique de la Paroi Bactérienne, 75015 Paris, France, bonecai@pasteur.fr.

Abstract

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the β-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro-N-acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via β-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc)5) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology.

KEYWORDS:

Neisseria; crystal structure; enzyme mechanism; hydrolase; lytic transglycosylase; peptidoglycan; structural biology

PMID:
29483188
PMCID:
PMC5912472
DOI:
10.1074/jbc.RA117.001095
[Indexed for MEDLINE]
Free PMC Article

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