As predicted through surface

topology analysis (CASTp), the groove volume at the active-site signature motifs of sDacD is 326.1 Ǻ3 (Fig. 2b), whereas that of sPBP5 is 960.8 Ǻ3 (Chowdhury & Ghosh, 2011). The smaller groove of sDacD possibly affects the binding of pentapeptide and, therefore, may decrease DD-CPase activity. However, activity toward smaller substrates such as Bocillin-FL may not be impaired. It is noteworthy that although the active-site groove volume of sDacD is nearly three times smaller than PBP5, it is about double the size of that of sPBP6 (161.5 Ǻ) (Chowdhury & Ghosh, 2011), which may explain why sDacD exerted better DD-CPase see more activity than sPBP6 towards pentapeptide substrate (Table 2). Unlike other DD-CPases, PBP5 mutant sensitizes E. coli to beta-lactam antibiotics and complementation of PBP5 restores the resistance (Sarkar et al., 2010). The reason for the PBP5-mediated beta-lactam resistance lies in its typical enzymatic properties. PBP5 deacylates beta-lactam more rapidly than PBP6 does (Chowdhury et al., 2010), even though PBP5 does not possess any beta-lactamase activity (Sarkar et al., 2010) at physiological pH, which is in disagreement with earlier claims (Georgopapadakou, 1993; Davies et al., 2001). It is proposed that PBP5 may behave as a trap for beta-lactams and provide a shielding effect over the lethal targets, which

in turn protects the essential PBPs from being inhibited (Sarkar et al., 2010). This may be due to the high deacylation efficiency and the high copy number of PBP5, and both factors taken together may act such that the effective pool of PF-01367338 mouse PBP5 remains available to bind beta-lactams. On the

other hand, PBP6 due to its low deacylation efficiency cannot reverse the lost beta-lactam resistance in PBP5 mutants, even when it is overexpressed (Sarkar et al., 2010, 2011). In contrast to PBP6, DacD can rescue the lost beta-lactam resistance in E. coli PBP5 mutant, at least partially (Sarkar et al., 2011). Our results reveal that sDacD possesses a higher rate of deacylation activity toward beta-lactams (~ 65% of PBP5) compared with PBP6. Therefore, it makes sense that DacD can partially substitute Protirelin the loss of PBP5 in terms of maintaining intrinsic beta-lactam resistance when expressed in mid-logarithmic phase. These observations imply that the cellular function of DacD is more closely related to PBP5 than with PBP6. In silico analyses of sDacD also reveals a possible structural relatedness with PBP5. Nevertheless, little differences in the orientation of the active-site residues exist, which probably cause these two proteins to act differently. The identical topology of sDacD and PBP5 at the Ω-type loop region predicts a high deacylation efficiency of sDacD. However, DacD possesses comparatively weak DD-CPase activity, possibly due to a far-reaching change in the orientation of Lys 46 from the active-site serine residue (Ser 43).