Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of B acillus spores

Abstract

A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi-redundant cortex-lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N-acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/β)8 -barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N-terminal peptidoglycan-binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL.

Stoichiometry, Absolute Abundance, and Localization of Proteins in the Bacillus cereus Spore Coat Insoluble Fraction Determined Using a QconCAT Approach

“…YpeB and SleB are known to interact 34 and appear to be present in a 5:4 ratio. YaaH is known to break down products of SleB and CwlJ 19 and is more abundant. It would be interesting to see if the combined copy numbers of SleB and CwlJ equal that of YaaH or if these enzymes are kinetically balanced instead.…”

“…Beyond determining the abundance, using a QconCAT also allows for determination of protein stoichiometry. Interplay between proteins is an important factor for acquisition of spore resistance mechanics, , and many spore proteins are known to interact with each other. − Information about protein stoichiometry can provide insight into how proteins relate to each other in vivo and give a sense of spatial dimensions occupied. Perturbations in this stoichiometry give a measure for integrity of the spore coat, which may be connected to acquired spore characteristics such as heat resistance or germination efficiency.…”

The deduced role of a chitinase containing two nonsynergistic catalytic domains

“…The crystal structures of chitinases with one single GH18 domain have been extensively studied in archaea (Tsuji et al, 2010), bacteria (Perrakis et al, 1994;van Aalten et al, 2000;Songsiriritthigul et al, 2008;Hsieh et al, 2010;Busby et al, 2012;Payne et al, 2012;Madhuprakash et al, 2013;Malecki et al, 2013;Ü stok et al, 2015;Itoh et al, 2016), fungi (Hollis et al, 2000;Rao et al, 2005;Hurtado-Guerrero & van Aalten, 2007;Schü ttelkopf et al, 2010;Yang et al, 2010), plants (Terwisscha van Scheltinga et al, 1994;Cavada et al, 2006;Ohnuma, Numata, Osawa, Mizuhara, Lampela et al, 2011;Ohnuma, Numata, Osawa, Mizuhara, Vå rum et al, 2011;Kitaoku et al, 2015;Masuda et al, 2015;Umemoto et al, 2015), insects Liu et al, 2017) and humans (Fusetti et al, 2002;Olland et al, 2009). Although the overall structure of these GH18 domains is a (/) 8 -barrel with a substrate-binding cleft on the top side, they can be differentiated by the shapes of the substrate-binding clefts and the presence or absence of insertion domains.…”

The crystal structure of Clostridium perfringens SleM, a muramidase involved in cortical hydrolysis during spore germination

“…The SleM crystal structure represents the first Clostridium CLE structure to be solved and the third CLE—after B. cereus and B. megaterium SleL—for which complete structures have been determined. Crystal structures for the catalytic domains of B. cereus and B. anthracis SleB, and B. subtilis YdhD (PDB‐ID: 3CZ8), have also been solved by X‐ray crystallography, meaning that of the major CLEs, three‐dimensional structural information is lacking only for CwlJ and SleC.…”

“…Regardless, the molecular mechanisms involved in the regulation of CLE catalytic activity in dormant and germinating spores, and the basis of CLE cortical substrate specificity, are poorly understood. Recent efforts to address these questions have employed X‐ray crystallography to reveal the three‐dimensional structures of several proteins involved in cortex hydrolysis . In addition to placing mechanistic studies of CLE activity and specificity on a solid structural footing, this information should enable a structure‐led approach to the design of inhibitors or stimulants of spore germination, which may in turn facilitate the development of novel therapeutic and decontamination strategies.…”