, 2002), and the mechanism by which it does so is probably related to its dd-CPase activity (Nelson et al., 2002; Ghosh this website & Young, 2003; Ghosh et al., 2008). However, E. coli also expresses PBP 6, which exhibits dd-CPase activity and is the most closely related homologue of PBP 5 (Goffin & Ghuysen, 1998; Ghosh et al., 2008). However, despite these resemblances, PBP 6 cannot substitute for PBP 5 in maintaining or restoring normal cell shape to PBP mutants (Nelson & Young, 2001; Nelson et al., 2002; Ghosh & Young, 2003). At least some of the relevant differences in the in vivo functions of these

two PBPs lie in a short stretch of residues in and near the active site (Ghosh & Young, 2003), but it is not known how Selleck Lumacaftor these sequence differences affect the enzymatic activities of these enzymes. Here, we investigated the kinetic properties of PBPs 5 and 6 and two mosaic proteins and found that the enzymes differ

in their substrate preferences and in the rates at which they remove the terminal d-alanine from these substrates. The results suggest that these differences correlate with the in vivo phenotypes of shape maintenance. PBP 5 is clearly a better dd-CPase than PBP 6. For example, depending on the substrate, the dd-CPase activity of PBP 5 was previously shown to be three to five times greater than that of PBP 6 (Amanuma & Strominger, 1980). In our assays, the dd-CPase activity of sPBP 5 was five times greater than that of sPBP 6 when tested against the substrate AcLAA. An even greater difference was observed when the enzymes were tested against the peptidoglycan mimetic substrate AGLAA, on which PBP 5 was active, but to which PBP 6 may not bind covalently or else it may bind, but may not cleave.

The failure of PBP 6 to act on this latter substrate is consistent with the observations of van der Linden et al. (1992). However, no dd-CPase activity was reported on AcLAA and UDP-muramyl pentapeptide Adenosine triphosphate substrates for either the membrane-bound or the soluble form of PBP 6 (van der Linden et al., 1992). We speculate that sPBP 6 exhibited a low level of dd-CPase activity toward the artificial substrate, AcLAA, possibly because the active site cleft of sPBP 6 might accommodate smaller substrates such as penicillin and AcLAA while being unable to bind a bulkier substrate such as AGLAA. The phenomenon of complete inertness of sPBP 6 toward the pentapeptide substrate is interesting in that it simultaneously raises a doubt as to whether it functions as dd-CPase in vivo at all. Previously, we found that the differences between PBPs 5 and 6 in complementing shape defects in vivo could be narrowed down to a short stretch of 20 contiguous residues within the active site (the MMD), where the two PBPs differ from one another by only seven amino acids (Ghosh & Young, 2003). Shape complementation was associated with the MMD from PBP 5 and not with that from PBP 6 (Ghosh & Young, 2003).