AcmA of Lactococcus lactis is an N‐acetylglucosaminidase with an optimal number of LysM domains for proper functioning

Abstract

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N‐terminal active site domain and a C‐terminal peptidoglycan‐binding domain. The active site domain is homologous to that of muramidase‐2 of Enterococcus hirae, however, RP‐HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N‐acetylglucosaminidase. In the C‐terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall‐hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

“…Proteins seem to possess a particular number of LysM domains for optimum PG binding and biological function. Both the addition and removal of LysM domains have resulted in decreases in PG-binding and enzymic activity (Shao et al, 2009;Steen et al, 2005). Loss of one or both LysM domains from SleL decreased the rate of PG hydrolysis as well as the affinity for cortex PG.…”

“…We suspect that the differences in digestion rates are likely indirect effects of decreases in the abilities of the protein derivatives to bind the substrate efficently. In bacterial hydrolases, LysM domains may be required to properly position the active site of a catalytic domain towards it substrate (Steen et al, 2005). Thus, in the absence of one or both LysM domains, digestion could still occur but would be solely dependent on substrate binding by the enzymic active site.…”

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

“…The LysM motif is a common module found in many cell-wall degrading enzymes and proteins involved in bacterial pathogenesis and is often present in multiple repeats [9][10][11]. It has been proposed that the LysM-type cell-wall binding domain binds non-covalently to peptidoglycan of various gram-positive bacteria [12].…”

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis

“…The autolysin AcmA is involved in cell separation and is the major effector of cellular autolysis in stationary phase (Buist et al, 1995). The AcmA protein has two domains: an N-terminal catalytic domain endowed with N-acetylglucosaminidase specificity (Steen et al, 2005a), and a C-terminal domain with three LysM modules involved in cell wall binding and which recognize peptidoglycan (Steen et al, 2003).…”