Dual Conserved Periplasmic Loops Possess Essential Charge Characteristics That Support a Catch-and-Release Mechanism of O-antigen Polymerization by Wzy in Pseudomonas aeruginosa PAO1

Abstract

Heteropolymeric B-band lipopolysaccharide in Pseudomonas aeruginosa PAO1 is synthesized via the so-called Wzy-dependent pathway, requiring a functional Wzy for polymerization of O-antigen repeat units in the periplasm. Wzy is an integral inner membrane protein for which the detailed topology has been mapped in a recent investigation (Islam, S. T., Taylor, V. L., Qi, M., and Lam, J. S. (2010) mBio 1, e00189-10), revealing two principal periplasmic loops (PL), PL3 and PL5, each containing an RX10G motif. Despite considerable sequence conservation between the two loops, the isoelectric point for each peptide displayed marked differences, with PL3 exhibiting a net-positive charge and PL5 showing a net-negative charge. Data from site-directed mutagenesis of amino acids in each PL have led to the identification of several key Arg residues within the two RX10G motifs that are important for Wzy function, of which Arg176, Arg290, and Arg291 could not be functionally substituted with Lys. These observations support the proposed role of each PL in a catch-and-release mechanism for Wzy-mediated O-antigen polymerization.

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

“…However, the Oag of Wzy Sf is neutral. Therefore, the charge property of the substrate for Wzy Sf is different from that of Wzy Pa .The RX 10 G motifs of PL3 and PL5 of Wzy Pa (20) are also absent in Wzy Sf . However, both PL3 and PL5 of Wzy Sf contained RX 15 G motifs (starting from R164 in PL3 and R289 in PL5) (Fig.…”

“…Modification of these residues (G176, D177, G323, and Y324) led to a loss of Oag polymerization (35). Islam et al showed that PL3 and PL5 of Wzy Pa have net positive and net negative charges, respectively, and they established their "catch-and-release" model (20). However, for Wzy Sf , we found that at a physiological pH both PL3 and PL5 possess net negative charges (the pI of PL3 is 4.65, and that of PL5 is 5.09).…”

“…Islam et al (20) performed extensive work on Pseudomonas aeruginosa Wzy (Wzy Pa ) and showed that both PL3 and PL5 of Wzy Pa contain RX 10 G motifs, which are important for the functioning of Wzy Pa . They also found that several Arg residues within these two motifs are also important for Wzy Pa function (20). However, there is little sequence identity between Wzy Pa and Wzy Sf , so it is not possible to predict the functional amino acid residues of Wzy Sf from another model.…”

Structural Characterization of Closely Related O-antigen Lipopolysaccharide (LPS) Chain Length Regulators

“…As subtle as the differences are, they would be sufficient to confer differential affinities to the O-antigen-PCP interaction. Recently, it was shown that the Wzy polymerase-O-antigen interactions in Pseudomonas are very sensitive to even conservative amino acid changes in specific positions as even Arg-to-Lys mutation led to a complete loss of the polymerase function, which was attributed to the loss of sugar binding (39). Clearly, the top of the molecule is not the only site of O-antigen-PCP interaction.…”

Enzymatic Transformations Involved in the Biosynthesis of Microbial Exo‐polysaccharides Based on the Assembly of Repeat Units

“…Onel oop is proposed to catch an ew repeat unit by using its net positive charge, and the second loop would function as retaining arm due to its net negative charge. [56] The substrate specificity of Wzy proteins has not been elucidated in detail, and only as mall number of gene-exchange or replacement experiments have been performed to obtain further insight in the acceptance of variousrepeat units. In general, observations of ap utative adaption mechanism of the Wzy proteins towards the length of the repeat units are described.…”