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 0006-2960

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Abstract

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
ISSN:	0006-2960
Date:	31 March 2009
Volume:	Vol.48
Number:	No.12
Number of Pages:	9
Page Range:	pp. 2675-2683
Identification Number:	10.1021/bi801993x
Status:	Peer Reviewed
Publication Status:	Published
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
URI:	http://wrap.warwick.ac.uk/id/eprint/28257

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