Pyridoxal phosphate (PLP) dependent enzymes are found in all three kingdoms of life and make up two percent of all enzymatic activity. From controlling steady state concentration of neuroactive amines (dopamine, GABA) and amino acids  (glutamate, glycine) to controlling the flux into important pathways involved in cell proliferation (polyamine pathways and the one carbon shuttle for thymine biosynthesis), this enzymatic class continues to be of considerable interest for biomedical science.Taking inspiration from Nature (e.g. vinylglycine and gabaculine) the Berkowitz lab has designed and enantioselectively synthesized a novel class of inhibitor candidates bearing a-vinylic functionalities. Of particular interest here, an inhibitor candidate containing a new fluorovinyl 'trigger', a-(2'Z-fluoro)vinyllysine (FVL), was stereoselectively synthesized in both D- and L-antipodal forms.  In this design, the a-(fluoro)vinylic branch is to serve as a masked warhead, to be specifically unmasked in the target enzyme active site (Trojan horse strategy).  In a quaternary context, this trigger is envisioned to be specific for PLP-AADCs (Amino Acid Decarboxylases) and retroaldolases, as no a-proton is present.  This �trigger� also incorporates a bioorthogonal fluorine substituent, designed to serve both a mechanistic and an analytical role, allowing the experimentalist to follow inactivation in the NMR tube.Lysine decarboxylase (LDC) from Hafnia alvei serves as a well-behaved model decarboxylase for the evaluation of this type of a-branched PLP enzyme inactivator candidate.  In fact, LDC is efficiently and enantioselectively inactivated by L-FVL.  Moreover, useful inhibitor partitioning information can be gleaned directly by following the inactivation via 19fluorine NMR spectroscopy.  X-ray crystallographic studies could potentially provide a final �nail in the coffin� in terms of molecular mechanism.  Toward this end, we report here the first structure of Hafnia alvei LDC and the use of this structure to model inhibitor chemistry.

Development of new PLP enzyme inactivators: From design to synthesis to evaluation, and now structural biology

248th National Meeting (2014)

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