Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus.
The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of approximately 5,800 M(-1) s(-1) and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.
Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie, B6a, B-4000 Sart-Tilman, Belgium.
Mohammed
M
Terrak
Martine
M
Nguyen-Distèche
D004789:Enzyme Activation; D016695:Glycosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium; D008345:Manganese; D016297:Mutagenesis, Site-Directed; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
D008345:Manganese; D016695:Glycosyltransferases; D008274:Magnesium