Peptidoglycan remodeling by the coordinated action of multispecific enzymes

Abstract

The peptidoglycan (PG) cell wall constitutes the main defense barrier of bacteria against environmental insults and acts as communication interface. The biochemistry of this macromolecule has been well characterized throughout the years but recent discoveries have unveiled its chemical plasticity under environmental stresses. Non-canonical D-amino acids (NCDAA) are produced and released to the extracellular media by diverse bacteria. Such molecules govern cell wall adaptation to challenging environments through their incorporation into the polymer, a widespread capability among bacteria that reveals the inherent catalytic plasticity of the enzymes involved in the cell wall metabolism. Here, we analyze the recent structural and biochemical characterization of Bsr, a new family of broad spectrum racemases able to generate a wide range of NCDAA. We also discuss the necessity of a coordinated action of PG multispecific enzymes to generate adequate levels of modification in the murein sacculus. Finally, we also highlight how this catalytic plasticity of NCDAA-incorporating enzymes has allowed the development of new revolutionary methodologies for the study of PG modes of growth and in vivo dynamics.

FIG. 1.

Crystal structure of the broad spectrum racemase (Bsr) of Vibrio cholerae. BsrV (PDB: 4BEU²⁴) is a homodimer with three special features that define its multispecificity: (i) it presents a wide entry channel that allows allocation of a diverse set of non-β-branching aliphatic and positively charged amino acids; (ii) the N-terminal extension of both monomers establishes polar interactions that improve the stability of the dimer and provokes a change in the disposition of the entry channels versus that observed for alanine racemase (Alr); (iii) and the catalytic machinery differs from that of Alrs as it presents a Cl⁻ ion (in green) instead of the typical N-carboxylated lysine, and several residues involved in pyridoxal-5-phosphate (PLP) coordination and substrate stabilization that constitute a molecular signature specific of this type of enzyme. AC, active center.

FIG. 2.

Pathways of non-canonical D-amino acids (NCDAA) incorporation in the cell wall of V. cholerae. (1) NCDAA can be acquired from the extracellular media or they can be produced extra-cytoplasmically by BsrV. (2) They can be incorporated into the fourth position of the peptide moiety by the action of LD-transpeptidases (Ldts). (3) ABC transporters provide LAA to the periplasmic BsrV and facilitate entrance of NCDAA, which can be used in the synthetic pathway of the peptidoglycan (PG) precursors. (4) Promiscuous Ddl and MurF ligases allow generation of NCDAA-modified Lipid II precursors. (5, 6) The synthetic penicillin-binding proteins (PBPs) polymerize and cross-link the PG through their glycosyltransferase (GT) and transpeptidase (TP) activities. Accumulation of modified pentapeptides depends on their DD-carboxypeptidase (DD-CP) or endopeptidase activities (EP) and their affinity to modified substrates. (7) Putative periplasmic LD-carboxypeptidase (LD-CP) activity could also account for the hydrolysis of modified tetrapeptides. OM, outer membrane; IM, inner membrane.