Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

Abstract

SummaryMycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactanpeptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an a/b hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles DlysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms

“…These two molecules are, therefore, the major candidates for substrates for the generation of FM, although cellular esterases that hydrolyze these have not been described. We note though that the mycobacteriophage lysin B enzyme has recently been shown to cleave mAGP (41), and there are several mycobacterial cutinase-like proteins for which lipolytic activity has been reported (42).…”

“…However, such enzymes may share common mechanisms with the mycolyltransferases, which use a catalytic serine (within a conserved GXSXG motif) and have conserved glutamine and histidine residues in the active site (47). We note that the recently described cutinase-like phage-encoded lysin B is also a serine esterase, with conserved serine, aspartic acid, and histidine residues in the active site, and cleaves mycolylarabinogalactan to release FM (41). In a search for related enzymes with potential serineesterase activity, we looked for ORFs annotated as serine esterase in the M. smegmatis genome web page (Comprehensive Microbial Resource at the J. Craig Venter Institute) and identified six ORFs (Fig.…”

Molecular Dissection of Phage Endolysin

“…The peptide was shown to switch between ␣-helix and ␤-sheet conformations by means of a Pro-Gly motif, depending upon the peptide concentration in micelles (26). The structure of Lysin B of mycobacteriophage D29 is available, and detailed experiments have been carried out to understand its activity (21). However, a similar level of characterization of Lysin A from the D29 phage has not yet been attempted.…”

“…Two of these genes encode Lysin A and Lysin B that sandwich one other gene coding for a membrane pore-forming protein, holin (20). The Lysin A (coded by gp10) is responsible for the hydrolysis of PG, 3 whereas Lysin B (coded by gp12) acts at the junction between mycolic acid and the PG (21,22). It should be noted here that the mycobacterial cell envelope contains, besides PG, an outer * The work was supported by a rapid grant from the Department of Biotechnology, Government of India.…”

Mycobacteriophage Marvin: a New Singleton Phage with an Unusual Genome Organization

“…The lysis cassette of Marvin is coded near the middle of the genome, a common location for mycobacteriophage genomes, and includes lysin A (gp51), the holin protein (gp54), and a putative lysin B (gp53) (16,42) (Fig. 3).…”