Endolysin CD27L causes cell lysis of the pathogen Clostridium difficile, a major cause of nosocomial infection. We report a structural and functional analysis of the catalytic activity of CD27L against C. difficile and other bacterial strains. We show that truncation of the endolysin to the N-terminal domain, CD27L 1-179 , gave an increased lytic activity against cells of C. difficile, while the C-terminal region, CD27L 180-270 , failed to produce lysis. CD27L 1-179 also has increased activity against other bacterial species that are targeted by the full-length protein and in addition was able to lyse some CD27L-insensitive strains. However, CD27L 1-179 retained a measure of specificity, failing to lyse a wide range of bacteria. The use of green fluorescent protein (GFP)-labeled proteins demonstrated that both CD27L and CD27L 1-179 bound to C. difficile cell walls. The crystal structure of CD27L 1-179 confirms that the enzyme is a zinc-dependent N-acetylmuramoyl-L-alanine amidase. A structure-based sequence analysis allowed us to identify four catalytic residues, a proton relay cascade, and a substrate binding pocket. A BLAST search shows that the closest-related amidases almost exclusively target Clostridia. This implied that the catalytic domain alone contained features that target a specific bacterial species. To test this hypothesis, we modified Leu 98 to a Trp residue which is found in an endolysin from a bacteriophage of Listeria monocytogenes (PlyPSA). This mutation in CD27L resulted in an increased activity against selected serotypes of L. monocytogenes, demonstrating the potential to tune the species specificity of the catalytic domain of an endolysin.

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.

Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.

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Arylamines constitute a large group of industrial chemicals detoxified by certain bacteria through conjugation reactions catalyzed by N -acetyltransferase (NAT) enzymes. NAT homologs, mostly from pathogenic bacteria, have been the subject of individual studies that do not facilitate direct comparisons. By implementing a practicable pipeline, we present comparative investigation of fifteen NAT homologs from ten bacteria, mainly bacilli, streptomycetes, and one alphaproteobacterium. The new homologs were characterized for their sequence, phylogeny, predicted structural features, substrate specificity, thermal stability, and interaction with components of the enzymatic reaction. Bacillus NATs demonstrated the characteristics of xenobiotic metabolizing N -acetyltransferases, with the majority of homologs generating high activities. Non-pathogenic bacilli are thus proposed as suitable mediators of arylamine bioremediation. Of the Streptomyces homologs, the NAT2 isoenzyme of S. venezuelae efficiently transformed highly toxic arylamines, while the remaining homologs were inactive or generated low activities suggesting that xenobiotic metabolism may not be their primary role. The functional divergence of Streptomyces NATs was consistent with their observed sequence, phylogenetic, and structural variability. These and previous findings support classification of microbial NATs into three groups. The first includes xenobiotic metabolizing enzymes with dual acetyl-/propionyl-CoA selectivity. Homologs of the second group are more rarely encountered, acting as malonyltransferases mediating specialized ecological interactions. Homologs of the third group effectively lack acyltransferase activity and their study may represent an interesting research area. Comparative NAT enzyme screens from a broad microbial spectrum may guide rational selection of homologs likely to share similar biological functions, allowing their combined investigation and use in biotechnological applications. IMPORTANCE Arylamines are encountered as industrial chemicals or byproducts of agrochemicals that may constitute highly toxic contaminants of soils and groundwaters. Although such chemicals may be recalcitrant to biotransformation, they can be enzymatically converted into less toxic forms by some bacteria. Therefore, exploitation of the arylamine detoxification capabilities of microorganisms is investigated as an effective approach for bioremediation. Among microbial biotransformations of arylamines, enzymatic conjugation reactions have been reported, including NAT-mediated N- acetylation. Comparative investigations of NAT enzymes across a range of microorganisms can be laborious and expensive, so here we present a streamlined methodology for implementing such work. We compare fifteen NAT homologs from non-pathogenic, free-living bacteria of potential biotechnological utility, mainly Terrabacteria known for their rich secondary and xenobiotic metabolism. The analysis allowed insights into the evolutionary and functional divergence of bacterial NAT homologs, combined with assessment of their fundamental structural and enzymatic differences and similarities.