Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coliO9a

Gregor Hagelueken,Bradley R Clarke,Tomas Lebl,Chris Whitfield,Hexian Huang,James H Naismith

doi:10.1111/mmi.12014

Abstract

SummaryThe Escherichia coli serotype O9a O-antigen polysaccharide (O-PS) is a model for glycan biosynthesis and export by the ATP-binding cassette transporter-dependent pathway. The polymannose O9a O-PS is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non-reducing mannose of the O-PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C-terminal coiled-coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho-associated protein kinase (ROCK). WbdD phosphorylates 2-α-d-mannosyl-d-mannose (2α-MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co-complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O-PS chain-length regulation in this important model system.

Highlights

Most Gram-negative bacteria contain lipopolysaccharide (LPS) as a major component of the outer membrane
The polymannose O9a O-antigen polysaccharide (O-PS) is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane
The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme

Summary

Introduction

Most Gram-negative bacteria contain lipopolysaccharide (LPS) as a major component of the outer membrane. The polymannose O-PSs of E. coli serotypes O8, O9 or O9a are prototypes for the ‘ATP transporter dependent’ pathway of O-PS synthesis (Fig. 1B). The length of the polymannose chain in E. coli O9 (and O9a) is controlled by a termination reaction, whereby a phosphate followed by a methyl group is added to the 3-OH position of the non-reducing terminal mannose residue of the chain (Clarke et al, 2004; 2011) (Fig. 1A). In E. coli O8 a methyl group is added to the 2-OH of the reducing mannose without phosphorylation (Vinogradov et al, 2002; Clarke et al, 2004) These modifications block further polymerization and are retained in the mature O-PS structure (Vinogradov et al, 2002; Kubler-Kielb et al, 2012). The O9/O9a WbdD homologue is a bifunctional kinase-methyltransferase, whereas the O8 WbdD is a Crystal structure of E. coli WbdD 731

Results

Discussion

Conclusion