Abstract
The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction.
Highlights
WaaL mediates the ligation of O-antigen onto lipid A-core
Of O-antigen repeating unit (O-unit)-PP-Lipid Substrates—Most of E. coli O-antigen polymers are ligated to Und-P using a GlcNAc or GalNAc reducing-end residue that is attached separately in the initial step(s) of the O-antigen biosynthesis
WaaL Exhibits Relaxed Specificity to Glycan Moiety of Donor Substrate—To further test whether the glycan moiety of the donor plays a critical role in the donor recognition by WaaL, we investigated the impact of variations in the glycan moiety on WaaL activity
Summary
WaaL mediates the ligation of O-antigen onto lipid A-core. Results: This ligation was reconstituted in vitro using synthetic donor substrates and donor mimics bearing structural variations. The LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Several functionally critical amino acid residues located in the large periplasmic loop or in the adjacent small periplasmic loop of these three WaaL homologues were identified [15,16,17] These critical residues, probably forming a part of a putative catalytic center that was suggested to participate in the chemical reaction(s) required for the release of O-antigen from the PP-Und lipid carrier [15], have been hypothesized to be involved in the binding of the pyrophosphate group of O-antigen-PP-Und [15, 16]. This assay enabled the elucidation of the specificity of WaaL toward the donor substrate as well as the identification of three functionally critical amino acid residues through the incorporation of mutant WaaL proteins into the assay
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