Enzymatic Synthesis of Putative Mycobacterial L,D‐Transpeptidase Substrates

Abstract

The Mycobacterium genus is diverse, with members ranging from harmless water or soil dwellers to the causative agent of the deadly disease tuberculosis. Bacterial cell wall biosynthetic enzymes are therapeutic targets, and mycobacterial cell walls are uniquely complex. The peptidoglycan (PG) layer of mycobacterial cell walls is largely and uniquely 3-3 crosslinked, and L,D-transpeptidase (Ldt) enzymes catalyze this transpeptidation reaction. Our lab has previously demonstrated that there are six mycobacterial Ldt classes, and the reason for this apparent redundancy is not well understood. The Mycobacterium tuberculosis (Mtb) class 2 Ldt, LdtMt2, is the most well studied, whereas the physiological roles of the remaining paralogs are largely unknown. The PG tetrapeptide L-Ala-D-isoGln-mesoDAPNH2-D-Ala is a known substrate for four of the five Mtb Ldts. While all mycobacterial species have at least five Ldt paralogs, only the Mtb enzymes LdtMt1, LdtMt2, LdtMt4, and LdtMt5 enzymes have confirmed 3-3 crosslinking activity. LdtMt3 does not crosslink this tetrapeptide substrate, and the transpeptidation capability of class 6 Ldts – of which there is no homolog in Mtb – have not been investigated. Orthologous Ldts are presumed to have the same activities, although this has not been biochemically confirmed, presumably because of limited access to putative substrates. Tetrapeptide (and many pentapeptide) PG substrates are not commercially available; accessing them is challenging because they contain unusual amino acids (D-isoGlu, D-isoGln, D-Ala, meso-diaminopimelic acid (mesoDAP), and amidated mesoDAP), and they are linked via the γ-carboxyl group of D-isoGln. Thus, even solid-support peptide synthesis is a challenge due to the requirement of non-commercial protected amino acids. LdtMt2 requires a fully amidated tetrapeptide substrate, yet 50% of the 3-3 crosslinked PG isolated from Mtb lacks amidation at the mesoDAP ε-carboxyl moiety of the donor stem peptide, suggesting at least one Mtb Ldt prefers a non-amidated donor substrate (or that amino groups are removed in subsequent biosynthetic steps). Further, some mycobacterial species, including Mycobacterium smegmatis, can utilize tetrapeptides with L-Lys in the third position. Thus, while the role of LdtMt2 in Mtb is known, the functions of the remaining paralogs and orthologous Ldts remain to be determined, highlighting the need for substrate specificity studies on these enzymes toward understanding their physiological roles. Here, we present our preliminary work in cloning and overexpressing PG biosynthetic enzymes toward characterizing the substrate preferences of mycobacterial Ldt enzymes. We are actively pursuing enzymatic synthesis of putative PG substrates and will be evaluating purified tetra- and pentapeptide substrates against the six classes of mycobacterial Ldt enzymes. The results from this study are expected to inform the community as to the physiological roles of the under studied Ldt paralogs.