Abstract 1840 Understanding the importance of two L,D-transpeptidases from Enterobacter cloacae through sequence alignments and Beta-lactam inhibition

L,D-transpeptidases (Ldts) as well as D,D-transpeptidases also known as penicillin binding proteins (PBPs) play an important role in formation of cross linkages in peptidoglycan, which is a key component of the cell wall. The functions of these two transpeptidases are similar, although their structures differ and Ldts are known to be a less dominant in most bacteria. Ldts, unlike PBPs, are not inhibited by most β-lactam antibiotics, however, carbapenems have been observed to inactivate them in some cases. This has led to the hypothesis that they are involved in resistance to β-lactam antibiotics. The role of Ldts in the antibiotic resistance of many pathogenic Gram-negative bacteria, such as Enterobacter cloacae, has not been studied. E. cloacae contains six hypothetical Ldt genes that are analogous to the six Ldts that found in E. coli. This study aims to investigate two of these Ldts found in E. cloacae, termed LdtD and LdtE, which are hypothesized to catalyze 3-3 cross-linkages based on their homology with E. coli proteins. To determine the closest homologs to E. cloacae LdtD and LdtE, we performed a protein sequence alignment with Ldts found in a wide range of bacterial species. This will also help to understand the evolutionary development of these two proteins. The sequence alignments of LdtE showed high conservation in the catalytic domain and more divergence in the N- and C-terminal domains. Going forward, sequence alignmentd for the other Ldts will be done to allow for a better understanding of when each Ldt evolved, which could give insight to their importance of their roll in cross-link formation. In addition, a panel of β-lactams were assayed to determine which demonstrates activity against LdtD and LdtE. A nitrocefin competition assay has been used to assay LdtE showing inhibition by meropenem, nafcillin, imipenem. Inactivation kinetics using the lambda-max of the β-lactam was also investigated for LdtD and LdtE. Our results have shown that the two carbapenems tested (imipenem and meropenem) are able to inhibit LdtE activity and no cephalosporins showed inhibition. LdtD is currently being investigated and assay conditions optimized to test for β-lactam inhibition. In conclusion, we have shown that the catalytic domain of LdtE in E. cloacae is highly conserved from a sequence alignment across over 20 bacterial species, but the N-terminus and C-terminus is variable. Using a nitrocefin competition and lambda-max reduction assays, the inhibition of LdtE and LdtD by various β-lactams was tested. We found that meropenem and imipenem, two carbapenems, and nafcillin, a penem, were able to inhibit LdtE. Funding for this research project was provided by The Ohio State University, College of Arts and Sciences, Division of Natural and Mathematical Sciences Seed Grant.