The capsule polymerase CslB of Neisseria meningitidis serogroup L catalyzes the synthesis of a complex trimeric repeating unit comprising glycosidic and phosphodiester linkages.
Neisseria meningitidis is a human pathogen causing bacterial meningitis and sepsis. The capsular polysaccharide surrounding N. meningitidis is a major virulence factor. The capsular polysaccharide consists of polyhexosamine phosphates in N. meningitidis serogroups A and X. The capsule polymerases (CPs) of these serogroups are members of the Stealth protein family comprising d-hexose-1-phosphate transferases from bacterial and protozoan pathogens. CslA, one of two putative CPs of the pathophysiologically less relevant N. meningitidis serogroup L, is one of the smallest known Stealth proteins and caught our attention for structure-function analyses. Because the N. meningitidis serogroup L capsule polymer consists of a trimeric repeating unit ([→3)-β-d-GlcNAc-(1→3)-β-d-GlcNAc-(1→3)-α-d-GlcNAc-(1→OPO3→]n), we speculated that the two predicted CPs (CslA and CslB) work together in polymer production. Consequently, both enzymes were cloned, overexpressed, and purified as recombinant proteins. Contrary to our expectation, enzymatic testing identified CslB to be sufficient to catalyze the synthesis of the complex trimeric N. meningitidis serogroup L capsule polymer repeating unit. No polymerase activity was detected for CslA, although the enzyme facilitated the hydrolysis of UDP-GlcNAc. Bioinformatics analyses identified two glycosyltransferase (GT) domains in CslB. The N-terminal domain modeled with 100% confidence onto a number of GT-A folded proteins, whereas the C-terminal domain modeled with 100% confidence onto TagF, a GT-B folded teichoic acid polymerase from Staphylococcus epidermidis. Amino acid positions known to have critical catalytic functions in the template proteins were conserved in CslB, and their point mutation abolished enzyme activity. CslB represents an enzyme of so far unique complexity regarding both the catalyzed reaction and enzyme architecture.
From the Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.
Christa
C
Litschko
Research, GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy, and.
Maria Rosaria
MR
Romano
Research, GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy, and.
Vittoria
V
Pinto
the Institute for Hygiene and Microbiology, University of Würzburg, 97080 Würzburg, Germany.
Heike
H
Claus
the Institute for Hygiene and Microbiology, University of Würzburg, 97080 Würzburg, Germany.
Ulrich
U
Vogel
Research, GSK Vaccines, Via Fiorentina 1, 53100 Siena, Italy, and.
Francesco
F
Berti
From the Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.
Rita
R
Gerardy-Schahn
From the Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany, Fiebig.Timm@mh-hannover.de.
Timm
T
Fiebig
D016667:Bacterial Capsules / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D019295:Computational Biology; D000939:Epitopes / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006027:Glycosides / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009345:Neisseria meningitidis / Q000737:chemistry*; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013212:Staphylococcus epidermidis; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry
D001426:Bacterial Proteins; D000939:Epitopes; D004952:Esters; D006027:Glycosides; D011108:Polymers; D011134:Polysaccharides; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C072175:CDP glycerol glycerophosphotransferase; D006026:Glycoside Hydrolases; C022562:capsular-polysaccharide galactohydrolase
CslB; Neisseria meningitidis; bifunctional enzyme; capsule polymerase; glycosyltransferase; hexose-1-phosphate transferase; high-performance liquid chromatography (HPLC); nuclear magnetic resonance (NMR); polysaccharide; recombinant protein expression