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Functional analysis of the large periplasmic loop of the Escherichia coli K‐12 WaaL O‐antigen ligase
Author(s) -
Pérez José M.,
McGarry Megan A.,
Marolda Cristina L.,
Valvano Miguel A.
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06490.x
Subject(s) - periplasmic space , biology , escherichia coli , biochemistry , amino acid , conserved sequence , peptide sequence , histidine , protein structure , mutant , sequence alignment , gene
Summary WaaL is a membrane enzyme implicated in ligating undecaprenyl‐diphosphate (Und‐PP)‐linked O antigen to lipid A‐core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K‐12 WaaL. Structural models of the EL5 from the K‐12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular α‐helices. One α‐helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non‐functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non‐conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und‐PP, which is the common component in all WaaL substrates.