STRUCTURE/FUNCTION OF PYRUVOYL AND PLP DEPENDENT ENZYMES

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The goal of this research is to understand the structure-function relationships and mechanisms of action of amino acid decarboxylases, a major class of pyridoxal phosphate (PLP)-dependent enzymes for which relatively little structural information is available. Decarboxylases are important therapeutic targets, generating biogenic amines such as histamine, dopamine, and polyamines. Structural information on decarboxylases and their inhibitor complexes are necessary to understand different PLP enzymes in sufficient detail to aid in the design of drugs targeted against their specific enzymatic activities. The X-ray structure, gene sequence and effector properties of a PLP- dependent ornithine decarboxylase (ODC) have been determined. We propose to use X-ray crystallography, supplemented with the techniques of site- directed mutagenesis and steady state kinetics, to examine the roles of key residues involved in the mechanism of action and effector activation of ODC from L.30a. Structures of inhibitor complexes will answer questions about "closed" and "open" forms of ODC and the nature and stereochemistry of catalytic intermediates. X-ray structures and kinetic properties of site-directed mutants will enable the assignment of specific roles to amino acid residues responsible for substrate specificity and binding, catalytic mechanism, GTP effector action, and subunit interactions. Sequence comparisons based on the structure of ODC from L.30a led us to suggest that there are at least two distinct structural families of decarboxylases. X-ray quality crystals have been produced for two examples of second class of decarboxylases, mouse ODC (which is very closely related to the human and trypanosomal ODCs) and biosynthetic arginine decarboxylase (bADC) from E. coli. The X-ray structures of these enzymes will be determined and mechanistic studies similar to those proposed for the ODC from L.30a will be extended to these systems. Crystals of mouse ODC-DFMO (difluoromethyl ornithine), a suicide inhibitor of ODC used in the treatment of African sleeping sickness, have also been obtained. These enzymes represent a new class of highly regulated, therapeutic targets for X-ray structural analysis. They also share a novel model of inactivation resulting from antizyme binding and conditions to produce decarboxylase-antizyme complex crystals will be screened.