Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus.
A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.
Wang
Q M
QM
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. qmwang@lilly.com
Peery
R B
RB
Johnson
R B
RB
Alborn
W E
WE
Yeh
W K
WK
Skatrud
P L
PL
D001426:Bacterial Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006090:Gram-Negative Bacteria / Q000201:enzymology; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009113:Muramidase; D010457:Peptidoglycan / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D001426:Bacterial Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C433790:MGT protein, Staphylococcus aureus; D009113:Muramidase