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Inhibition studies of glu ligase TTLL7 and peptidoglycan peptidase Csd4

University of British Columbia

Microtubules are one of the major components of the cytoskeleton, and are comprised of two kinds of tubulin proteins. The presence of diverse post-translational modifications provides microtubules with multiple biological functions, including both structural and physiological roles. Polyglutamylation is one of the widespread post-translational modifications and is catalyzed by the tubulin tyrosine ligase-like (TTLL) enzymes. The reaction adds multiple glutamates to the glutamate side chain near the tubulin C-terminus to form a polyglutamate chain of various lengths. Such a side chain can be recognized by microtubule-associated proteins (MAPs) as a regulation signal. Our work focuses on the inhibition studies of the enzyme TTLL7. Three inhibitors (Inhibitor 1 – 3) targeting different stages of polyglutamylation were designed and synthesized. The inhibitors were then tested in our collaborator Dr. Roll-Mecak’s Lab, and we found Inhibitor 2, that targets an elongation process, had the highest potency with an IC₅₀ of approximately 150 μM. Peptidoglycan is a key component of the bacterial cell wall and plays an essential role in determining bacterial cell morphology. The cell shape determining genes were recently discovered in Helicobacter pylori, which encode different DD-, DL- or endo-, carboxypeptidases. Theses peptidases hydrolyze the peptide bond in either crosslinked or uncrosslinked muropeptides in order to alter bacterial cell shape. Our study focuses on the enzyme Csd4, a carboxypeptidase that cleaves the PG tripeptides PG-L-Ala-iso-D-Glu-meso-Dap to the dipeptide PG-L-Ala-iso-D-Glu. Its homolog Pgp1 in Campylobacter jejuni was also identified recently. We synthesized a simple tripeptide Ac-L-Ala- iso-D-Glu-meso-Dap as a substrate for Csd4, which was successfully co-crystallized with the enzyme. We also measured the activity of Csd4 with this substrate. The Michaelis constant (KM) and catalytic rate constant (kcat) are 112 μM and 0.044 s-¹, respectively. Based on a mechanistic analysis, we designed and synthesized a pseudodipeptidyl phosphinate as a Csd4 inhibitor. The inhibitor was tested and gave a KI of 3.3 μM. The crystal structure of Csd4-inhibitor proved the supported mechanism involving ligation of the oxyanion tetrahedral intermediate by the zinc ion. In vivo studies with the inhibitor showed it induced significant cell straightening in H. pylori and acapsular C. jejuni at millimolar concentration.

Text

2015-07-21

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54127

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/