Examining Differences in the Binding Sites of Succinyl Transferases DapD and TabB

Abstract

TabB and DapD are N‐succinyl transferases that succinylate tetrahydrodipicolinic acid (THDPA) during the biosynthesis of the natural product tabtoxin and lysine, respectively. Although TabB and DapD catalyze identical reactions, and are predicted to form homologous, trimeric left‐handed β‐helical structures, close examination of their sequences reveal that the three‐dimensional arrangements of functionality within their respective active sites diverge significantly. Further examination of the sequences and modeled structures of DapD and TabB suggest that they are representatives of distinct classes of THDPA N‐succinyl transferases, with a small number of amino acids that appear to distinguish “TabB‐class” from “DapD‐class” enzymes. The goal of this work was to test whether it was possible to “swap” one active site for the other within a single three‐dimensional scaffold. In total, we created sixteen DapD and TabB mutants. Four swapped amino acids in DapD for amino acids in TabB and vice versa with the goal of morphing one binding site into the other. The other twelve mutations replaced one amino acid for other similar, yet structurally distinct amino acids. The mutated plasmids were then transformed into E. coli and the corresponding TabB and DapD mutants were overexpressed and isolated. We tested the activities of these mutants via a colorimetric Ellman assay that detected the formation of the reaction coproduct CoASH. Initial results suggest that there is some plasticity within the TabB and DapD active sites, and future efforts are directed to exploring the boundaries of this catalytic flexibility.