TY - JOUR
T1 - Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli
AU - Terrak, M.
AU - Sauvage, E.
AU - Derouaux, A.
AU - Dehareng, D.
AU - Bouhss, A.
AU - Breukink, E.J.
AU - Jeanjean, S.
AU - Nguyen-Distèche, M.
PY - 2008
Y1 - 2008
N2 - The peptidoglycan glycosyltransferase (GT) module of class A
penicillin-binding proteins (PBPs) and monofunctional GTs catalyze
glycan chain elongation of the bacterial cell wall. These
enzymes belong to the GT51 family, are characterized by five conserved
motifs, and have some fold similarity with the phage
lysozyme. In this work,wehave systematically modified all the conserved
amino acid residues of the GT module of Escherichia coli
class A PBP1b by site-directed mutagenesis and determined their
importance for the in vivo and in vitro activity and the thermostability
of the protein. To get an insight into theGTactive site of this
paradigm enzyme, a model of PBP1b GT domain was constructed
based on the available crystal structures (PDB codes 2OLV and
2OLU). The data show that in addition to the essential glutamate
residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237
and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif
2, all located in the catalytic cavity of the GT domain, are essential
for the in vitro enzymatic activity of the PBP1b and for its in vivo
functioning. Thus, the first three conserved motifs contain most of
the residues that are required for theGTactivity of the PBP1b. The
residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to
maintain the structure of the active site and the positioning of the
catalytic Glu233.
AB - The peptidoglycan glycosyltransferase (GT) module of class A
penicillin-binding proteins (PBPs) and monofunctional GTs catalyze
glycan chain elongation of the bacterial cell wall. These
enzymes belong to the GT51 family, are characterized by five conserved
motifs, and have some fold similarity with the phage
lysozyme. In this work,wehave systematically modified all the conserved
amino acid residues of the GT module of Escherichia coli
class A PBP1b by site-directed mutagenesis and determined their
importance for the in vivo and in vitro activity and the thermostability
of the protein. To get an insight into theGTactive site of this
paradigm enzyme, a model of PBP1b GT domain was constructed
based on the available crystal structures (PDB codes 2OLV and
2OLU). The data show that in addition to the essential glutamate
residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237
and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif
2, all located in the catalytic cavity of the GT domain, are essential
for the in vitro enzymatic activity of the PBP1b and for its in vivo
functioning. Thus, the first three conserved motifs contain most of
the residues that are required for theGTactivity of the PBP1b. The
residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to
maintain the structure of the active site and the positioning of the
catalytic Glu233.
M3 - Article
SN - 0021-9258
VL - 283
SP - 28464
EP - 28470
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 42
ER -