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The emergence of bacterial strains that are resistant to virtually all currently available antibiotics underscores the importance of developing new antimicrobial compounds. N succinyl LL diaminopimelic acid desuccinylase (DapE) is a metallohydrolase involved in the meso diaminopimelate (mDAP)/lysine biosynthetic pathway necessary for lysine biosynthesis and for building the peptidoglycan cell wall. Because DapE is essential for Gram negative and some Gram positive bacteria DapE has been proposed as a good target for antibiotic development. Recently L captopril has been suggested as a lead compound for inhibition of DapE although its selectivity for this enzyme target in bacteria remains unclear (Gillner et al. (2009)). Here we tested the selectivity of L captopril against DapE in bacteria. Since DapE knockout strains of gram negative bacteria are viable upon chemical supplementation with mDAP we reasoned that the antimicrobial activity of compounds targeting DapE should be abolished in mDAP containing media. Although L captopril had modest antimicrobial activity in Escherichia coli and in Salmonella enterica to our surprise inhibition of bacterial growth was independent both of mDAP supplementation and DapE over expression. We conclude that DapE is not the main target of L captopril inhibition in these bacteria. The methods implemented here will be useful for screening DapE selective antimicrobial compounds directly in bacterial cultures.

Enzyme promiscuity, defined as functional properties other than those for which they are evolved, is considered a key factor in the evolution of new enzyme functions. Many metalloproteins can be alternatively metallated, which may lead to metal-dependent promiscuity. The mechanisms and evolutionary implications of metal-mediated promiscuity appear to be underexplored, especially considering that approximately one-third of structurally characterized proteins are thought to be metalloproteins. Here, we investigated the bacterial binuclear metallohydrolase, N-Succinyl-L-LDiaminopimelic acid desuccinylase DapE (EC 3.5.1.18) of S.enterica. DapE is an essential enzyme in the late stage of the lysine biosynthetic pathway that also provides a crucial building block of the peptidoglycan cell wall. Since DapE is essential for most Gram-negative and many Gram-positive bacteria and it is not present in humans, it has been proposed as a very good target for antibiotic development. It was also reported that DapE has a metal dependent promiscuous aspartyl dipeptidase activity, in which incorporation of either zinc or manganese to the enzyme leads to activity with different substrates and this phenomenon occurs both in vivo and in vitro. We addressed the reaction mechanism of the native desuccinylase activity as well as the Mn2+-dependent aspartyl dipeptidase promiscuous activity of DapE, by investigating a series of substrate analogues and potential inhibitors. We postulated a plausible mechanism for metal-dependent promiscuity, based on subtle differences in coordination preferences between Mn2+ and Zn2+,which may be widely applicable to other enzymes. We revealed why a promising inhibitor of the enzyme in vitro, L-captopril, fails to exert antibiotic activity and propose broad practical implications of this discovery for drug-design, as well as fundamental evolutionary implications. We described kinetic cooperativity in DapE, which offers clues on structural rearrangements that occur during catalysis and is also of relevance to inhibitor design. Finally, we explored the evolutionary aspects of functional robustness of native activity over the promiscuous activity using DapE as a model enzyme and addressed the molecular mechanisms underlying the emergence of functional robustness through laboratory evolution.