CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation.
The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.
Cartee
Robert T
RT
Department of Microbiology, BBRB 661/12, 845 19th St. South, Birmingham, AL 35294, USA.
Forsee
W Thomas
WT
Bender
Matthew H
MH
Ambrose
Karita D
KD
Yother
Janet
J
D016667:Bacterial Capsules / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005958:Glucosephosphates / Q000378:metabolism*; D006017:Glycolipids / Q000032:analysis; D008969:Molecular Sequence Data; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D001426:Bacterial Proteins; D005958:Glucosephosphates; D006017:Glycolipids; D011103:Polyisoprenyl Phosphate Monosaccharides; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; C011082:polyprenyl monophosphate; C068691:polyprenyl-phosphate glucose; C031590:glucose-1-phosphate; D014532:Uridine Diphosphate Glucose