GNA33 from Neisseria meningitidis serogroup B encodes a membrane-bound lytic transglycosylase (MltA).
In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48-kDa lipoprotein that is 34.5% identical with membrane-bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation-exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6-anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 degrees C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X-100 stimulated activity 5.1-fold. Affinity chromatography indicated that MltA interacts with penicillin-binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.
Jennings
Gary T
GT
I.R.I.S., Chiron S.p.A., Siena, Italy. jennings@cytos.com
Savino
Silvana
S
Marchetti
Elisa
E
Aricò
Beatrice
B
Kast
Thomas
T
Baldi
Lucia
L
Ursinus
Astrid
A
Höltje
Joachim-Volker
JV
Nicholas
Robert A
RA
Rappuoli
Rino
R
Grandi
Guido
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D009345:Neisseria meningitidis / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D010457:Peptidoglycan / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012703:Serotyping; D013379:Substrate Specificity
D008074:Lipoproteins; D008565:Membrane Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D016695:Glycosyltransferases; C085441:MltA protein, bacteria; D009113:Muramidase