A product of RpfB and RipA joint enzymatic action promotes the resuscitation of dormant mycobacteria

Abstract

Resuscitation-promoting factor proteins (Rpfs) are known to participate in reactivating the dormant forms of actinobacteria. Structural analysis of the Rpf catalytic domain demonstrates its similarity to lysozyme and to lytic transglycosylases -the groups of enzymes that cleave the b-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc, and concomitantly form a 1,6-anhydro ring at the MurNAc residue. Analysis of the products formed from mycobacterial peptidoglycan hydrolysis reactions containing a mixture of RpfB and resuscitation-promoting factor interacting protein (RipA) allowed us to identify the suggested product of their action -N-acetylglucosaminyl-b(1?4)-N-glycolyl-1,6-anhydromuramyl-Lalanyl-D-isoglutamate. To identify the role of this resulting product in resuscitation, we used a synthetic 1,6-anhydrodisaccharide-dipeptide, and tested its ability to stimulate resuscitation by using the dormant Mycobacterium smegmatis model. It was found that the disaccharide-dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9-100 ngÁmL À1 . The current study therefore provides the first insights into the molecular mechanism of resuscitation from dormancy involving a product of RpfB/RipA-mediated peptidoglycan cleavage.

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off‐the‐wall Balance of Synthesis and Degradation

“…Consistent with these findings, we previously showed that RpfB acts through a mechanism, which resembles that of lytic transglycosylases, rather than that of lysozyme . Consistently, the product of the joint action of RpfB and RipA, is the anhydro‐muropeptide N ‐acetylglucosaminyl‐(ß1→4)‐ N ‐acetyl‐1,6‐anhydromuramoyl‐ l ‐alanyl‐ d ‐isoglutamate (anhydroGMDP) . Similar to other lytic transglycosylases, RpfB cleaves the β‐1, 4‐glycosidic bond between NAM and NAG with the formation of a 1,6‐anhydro ring at the NAM residue.…”

“…In contrast to HEWL, RpfB has no equivalent to c‐type lysozyme's Asp52, and a sole extra acidic amino acid in the substrate binding cleft, Asp312, exists only in RpfB, and is not conserved in its M. tuberculosis homologs. Consistent with these findings, we previously showed that RpfB acts through a mechanism, which resembles that of lytic transglycosylases, rather than that of lysozyme . Consistently, the product of the joint action of RpfB and RipA, is the anhydro‐muropeptide N ‐acetylglucosaminyl‐(ß1→4)‐ N ‐acetyl‐1,6‐anhydromuramoyl‐ l ‐alanyl‐ d ‐isoglutamate (anhydroGMDP) .…”

“…Despite sharing a low sequence identity, the RpfB catalytic domain exhibits structural similarity to lysozyme, an enzyme for which the mechanism is well and thoroughly studied . Consistent with structural studies, RpfB is predicted to cleave the glycosidic bond between N ‐acetylglucosamine ( N ‐AcGlc) and N ‐acetylmuramic ( N ‐Ac‐Mur) acid in PGN of mycobacterial cell wall –. In hen egg white lysozyme (HEWL), two residues have been found to be critical for enzyme activity, Glu35 and Asp52.…”

“…Based on these considerations, all extra domains of RpfB seem to be functional to improve the protein interactions with PGN and to properly orient the catalytic site cleft of RpfB on the glycan chains of PGN. Crystallographic and MD simulations on the complexes between RpfB and NAG3/NAG6 along with the analysis of the products formed as a result of PGN hydrolysis reaction provided several clues to understanding of the mechanism of this reaction . A NAG6 sugar chain saturates all six potential binding subsites (−4 to +2) of the enzyme.…”

“…The interaction between RipA and RpfB results in synergetic hydrolysis of mycobacterial PGN because the cooperativity may either be associated with the allosteric activation of one of these proteins or to their enhanced ability to release free muropeptides by acting both on the glycan and peptide moieties . In addition, the question of how PGN hydrolysis is bound with the reactivation of the dormant cells remains to be elucidated.…”