The L,D-carboxypeptidase DacB plays a key role in the remodelling of Streptococcus pneumoniae peptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB from S. pneumoniae TIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB from S. pneumoniae D39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, with Km and kcat values of 2.84 ± 0.37 mM and 91.49 ± 0.05 s(-1), respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.
Keywords: Streptococcus pneumoniae; l,d-carboxypeptidase; molecular docking; peptidoglycan.