Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial Arabinogalactan.
Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C(4)-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.
Alberta Glycomics Centre, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Robert W
RW
Wheatley
Ruixiang Blake
RB
Zheng
Michele R
MR
Richards
Todd L
TL
Lowary
Kenneth K S
KK
Ng
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D005685:Galactans; D005690:Galactose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005685:Galactans; C104136:uridine diphosphate galactofuranose; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; C430543:Rv3808c protein, Mycobacterium tuberculosis; C005653:arabinogalactan; D005690:Galactose