Mutational analysis of the substrate specificity of Escherichia coli penicillin binding protein 4
Clarke, Thomas B., Kawai, Fumihiro, Park, Sam-Yong, Tame, Jeremy R. H., Dowson, Christopher G. and Roper, David I.. (2009) Mutational analysis of the substrate specificity of Escherichia coli penicillin binding protein 4. Biochemistry, Vol.48 (No.12). pp. 2675-2683. ISSN 0006-2960Full text not available from this repository.
Official URL: http://dx.doi.org/10.1021/bi801993x
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 14 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 (Asp 155, 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 Asp 155, 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.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry|
|Divisions:||Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)|
|Journal or Publication Title:||Biochemistry|
|Publisher:||American Chemical Society|
|Official Date:||31 March 2009|
|Number of Pages:||9|
|Page Range:||pp. 2675-2683|
|Access rights to Published version:||Restricted or Subscription Access|
|Funder:||Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Medical Research Council (Great Britain) (MRC)|
|Grant number:||BBSSC200412641 (BBSRC), G0400848, G500643|
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