Cell-wall turnover, an enzymatic process that results in the loss of peptidoglycan (PG) components, has been reported in many bacteria, including Escherichia coli
and Bacillus subtilis
(Doyle et al.
). The products of the turnover are generally re-utilized through a process known as PG recycling (Park & Uehara, 2008
). The molecular processes involved in cell-wall turnover and recycling are not currently well understood in comparison to cell-wall synthesis, particularly in bacteria other than E. coli
(Park & Uehara, 2008
; Uehara & Park, 2003
; Uehara et al.
). In E. coli
, the cell wall is degraded by lytic transglycosylases that release anhydromuropeptides (GlcNAc-anhMurNAc-l
-Ala, where DAP is meso
-diaminopimelic acid), which are imported into the cytoplasm, primarily by AmpG permease, and subsequently processed by N
-alanine amidase (which cleaves between GlcNAc-anhMurNAc and l
-Ala) and ld
-carboxypeptidase LdcA (which cleaves between DAP and d
-Ala). Two fates are possible for the generated murein tripeptide l
-Glu-DAP. Under normal growth conditions, these tripeptides are recycled by Mpl ligase and returned to the peptidoglycan-biosynthetic pathway. During nutrient-limiting conditions, an additional pathway (Fig. 1) is likely to be involved in the murein tripeptide metabolism, as proposed in E. coli
(Uehara & Park, 2003
). MpaA endopeptidase, a metallocarboxypeptidase, specifically cleaves l
-Glu-DAP to produce l
-Glu and DAP. l
-Glu is then converted to l
-Glu and subsequently to l
-Ala and l
-Glu by YcjG epimerase and PepD peptidase, respectively.
Proposed metabolic pathways for murein peptides in E. coli and B. subtilis.
Some of the enzymes in the cell-wall recycling of E. coli
, such as AmpG, AmpD, Mpl and MpaA, have no orthologs in B. subtilis
(Park & Uehara, 2008
), suggesting that the mechanism of cell-wall recycling may differ between the two bacteria. In B. subtilis
, PG was proposed to be cleaved by a muramidase and an amidase to produce GlcNAc-MurNAc and stem peptides (Park & Uehara, 2008
). The free peptides are imported into the cytoplasm by an unidentified permease and subsequently processed by YkfABC enzymes. YkfA, an ld
-carboxypeptidase, removes the terminal d
-Ala. The generated tripeptide is further metabolized by a pathway that is functionally equivalent to that of E. coli
, with YkfC as the γ-d
-Glu-DAP endopeptidase and YkfB as the l
-Glu epimerase (Fig. 1) (Schmidt et al.
). Interestingly, while YkfB is homologous to YcjG of E. coli
, YkfC is unrelated to MpaA in sequence and structure despite having an equivalent function.
YkfC contains a C-terminal NlpC/P60 cysteine peptidase domain. NlpC/P60 is a large family of cell-wall related cysteine peptidases that are broadly distributed in bacteria, viruses, archaea and eukaryotes (Anantharaman & Aravind, 2003
; Bateman & Rawlings, 2003
; Rigden et al.
). Characterized NlpC/P60 enzymes are almost all γ-d
-Glu-DAP (or γ-d
-Glu-Lys) endopeptidases. While their biochemical function seems to be conserved, the physiological roles of NlpC/P60 proteins are diverse, including involvement in cell separation, expansion, differentiation, cell-wall turnover, cell lysis, protein secretion and virus infection (Smith et al.
). Secreted NlpC/P60 proteins also have other roles in pathogenesis. The autolysin P60 of Listeria monocytogenes
is involved in host-cell invasion (Kuhn & Goebel, 1989
), enterotoxin FM of B. cereus
in food poisoning (Asano et al.
), and SagA of Enterococcus faecium
is a secreted antigen that binds to extracellular matrix proteins (Teng et al.
NlpC/P60 proteins can be lethal to bacteria owing to their ability to compromise cell-wall integrity or cell-wall biosynthesis. Therefore, their activities are tightly controlled through multiple mechanisms (Smith et al.
); their expression is regulated at the transcription level and their cellular localization is dependent on their physiological roles. Furthermore, their atomic structures are highly optimized to precisely define their substrate specificity (Xu et al.
). NlpC/P60 proteins are often fused to auxiliary domains, many of which are known cell-wall binding modules (e.g.
LysM and the choline-binding domain). Thus, it is generally assumed that these auxiliary domains function as targeting domains which localize their proteins to the cell wall. The functional synergy between the NlpC/P60 domains and their auxiliary domains is currently not fully understood. We have previously determined the crystal structure of a γ-d
-Glu-DAP endopeptidase from cyanobacteria (AvPCP/NpPCP; Anabaena variabilis
PG cysteine peptidase; Xu et al.
) and showed that it contained an N-terminal bacterial SH3 (SH3b) domain and a C-terminal NlpC/P60 domain. We proposed that the SH3b domain of this enzyme is important in defining the substrate specificity of the peptidase domain. However, the mechanism of substrate recognition by NlpC/P60 and SH3b was not firmly established. Here, we report the crystal structure of YkfC from B. cereus
(BcYkfC) in complex with l
-Glu. BcYkfC shares 40% sequence identity with YkfC from B. subtilis
, which has previously been biochemically characterized (Schmidt et al.
). Thus, we now have the first detailed view of substrate recognition by an NlpC/P60 protein.