Functional characterization of domains found within a lytic enzyme produced by Streptococcus equi subsp. zooepidemicus

Abstract

Zoocin A is a lysostaphin-like streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that specifically targets the cell walls of some closely related species. On the basis of sequence homology it was suggested that zoocin A was a domainstructured enzyme with the N-terminal domain responsible for catalysis (CAT) and the C-terminal domain for target recognition (SBD). Polypeptides corresponding to zoocin A (rZooA) and each of the putative domains (rCAT and rSBD) were prepared by use of recombinant technology. The biological activities of each was compared by use of a dye-release assay and a cell-binding assay. Cell wall hydrolysis was shown to be a function of CAT and target recognition a function of the SBD. Expression of the zoocin A immunity factor gene produced cell walls resistant to hydrolysis by either rZooA or its component domains, and with reduced capacity to bind rZooA and rSBD.

The metal binding site of zoocin A

“…Streptococcolytic activity of rZOO and Cd-rZOO was determined by use of a dye-release assay with Remazol brilliant blue-labeled, heat-killed cells of Streptococcus pyogenes FF22 as substrate as previously described [7].…”

“…The protein is composed of two domains, an N-terminal catalytic domain (CAT) followed by a threonine-proline rich linker sequence, and then the target-recognition domain (TRD), which is responsible for specific recognition of cell wall architecture on sensitive strains [7]. Zoocin is a member of a superfamily of endopeptidases that selectively hydrolyze peptide cross bridges in peptidoglycans of the cell wall.…”

“…The catalytic region of LytM shows homology to both zoocin (35% identities, 54% positive matches by BLAST 2 alignment) and lysostaphin (48% identities, 63% positive matches). Zoocin has been over-expressed and purified from Escherichia coli (rZOO) as have the catalytic (rCAT) and target-recognition (rTRD) domains, each with only half of the putative TP-linkers and all with an N-terminal MRGS(H) 6 hexahistidine tag for Ni-affinity column purification [7].…”

Enzybiotics and their Potential Applications in Medicine

“…zooepidemicus 4881 (Akesson et al 2007 ). It is composed of an N -terminal catalytic domain of putative endopeptidase activity and a C -terminal cell wall -binding domain (Lai et al 2002 ). It was shown that zoocin A -susceptible streptococcal species include, aside from S. equi , S. pyogenes , Streptococcus mutans , and S. gordonii .…”

Produção de bacteriocina por Bifidobacterium lactis a partir de soro de leite

“…Their action mechanism is different from those of other bacteriocins, in that they promote lysis of the cell wall of the target microorganism. Their Nterminal portion is homologous to an endopeptidase involved in cell wall synthesis, while the C-terminal portion is responsible for recognition of the target cell (Lai, Tran, & Simmonds, 2002).…”