Imperial College London


Faculty of MedicineDepartment of Medicine

Research Fellow



+44 (0)20 7594 2074thomas.clarke




5.40CFlowers buildingSouth Kensington Campus






BibTex format

author = {Clarke, TB and Kawai, F and Park, S-Y and Tame, JRH and Dowson, CG and Roper, DI},
doi = {10.1021/bi801993x},
journal = {Biochemistry},
pages = {2675--2683},
title = {Mutational analysis of the substrate specificity of Escherichia coli penicillin binding protein 4.},
url = {},
volume = {48},
year = {2009}

RIS format (EndNote, RefMan)

AB - Escherichia coli PBP4 is the archetypal class C, low molecular mass penicillin binding protein (LMM-PBP) and possesses both dd-carboxypeptidase and dd-endopeptidase activity. In contrast to other classes of PBP, class C LMM-PBPs show high dd-carboxypeptidase activity and rapidly hydrolyze synthetic fragments of peptidoglycan. The recently solved X-ray crystal structures of three class C LMM-PBPs (E. coli PBP4, Bacillus subtilis PBP4a, and Actinomadura R39 dd-peptidase) have identified several residues that form a pocket in the active site unique to this class of PBP. The X-ray cocrystal structure of the Actinomadura R39 DD-peptidase with a cephalosporin bearing a peptidoglycan-mimetic side chain showed that residues of this pocket interact with the third position meso-2,6-diaminopimelic acid residue of the peptidoglycan stem peptide. Equivalent residues of E. coli PBP4 (Asp155, Phe160, Arg361, and Gln422) were mutated, and the effect on both DD-carboxypeptidase and DD-endopeptidase activities was determined. Using N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine as substrate, mutation of Asp155, Phe160, Arg361, and Gln422 to alanine reduced k(cat)/K(m) by 12.7-, 1.9-, 24.5-, and 13.8-fold, respectively. None of the k(cat) values deviated significantly from wild-type PBP4. PBP4 DD-endopeptidase activity was also affected, with substitution of Asp155, Arg361, and Gln422 reducing specific activity by 22%, 56%, and 40%, respectively. This provides the first direct demonstration of the importance of residues forming a subsite to accommodate meso-2,6-diaminopimelic acid in both the DD-carboxypeptidase and DD-endopeptidase activities of a class C LMM-PBP.
AU - Clarke,TB
AU - Kawai,F
AU - Park,S-Y
AU - Tame,JRH
AU - Dowson,CG
AU - Roper,DI
DO - 10.1021/bi801993x
EP - 2683
PY - 2009///
SP - 2675
TI - Mutational analysis of the substrate specificity of Escherichia coli penicillin binding protein 4.
T2 - Biochemistry
UR -
UR -
VL - 48
ER -