OMP decarboxylase : preparation of purified protein inhibitors for X-ray crystallography and NMR

Abstract

OMP decarboxylase is the final enzyme of the de novo pyrimidine biosynthetic pathway and it displays one of the largest rate enhancements ever measured. The mechanism by which the enzyme carries out decarboxylation remains unknown. Several mechanism have been proposed to explain the activity of ODCase. Our group tried to support the mechanism involving Lys93 protonation of O2 by crystallographic and NMR studies using the inhibitor, UMP-6-thiocarboxamide (UMP-CSNH2), a substrate isostere. The E. coli ODCase gene was cloned for overproduction of protein, and the resulting ODCse was purified by applying to the Affi-Gel Blue column and eluting using different inhibitors. The inhibitor-free protein used for different protein/inhibitor samples was obtained by eluting protein from the column with UMP, and dialyzing the pooled fractions three times. Unenriched and 15N-enriched UMP-CSNH2 were synthesized and purified, for the use in the preparation of ODCase-inhibitor samples for NMR and crystallography. The bond distances of hydrogens attached to 15N and hydrogens attached to C5' were measured using molecular modeling, in order to determine whether or not NMR signals resulting from close proximity of these hydrogens is possible. An attempt was made to synthesize BMP, a potent ODCase inhibitor, by enzymatic catalysis using OPRTase. Different protein and protein inhibitor samples were prepared for crystallographic and NMR studies. Crystallization of ODCase with UMP-CSNH2, carried out at the University of Toledo, appears promising.