(759b) A Tale of Two Enzyme Folds: Insights Into Catalysis by R67 and E. Coli Dihydrofolate

The amino acid residual motions are primarily responsible for a variety of functions including protein folding and enzymatic reactions. Enzymatic reactions prove to extremely efficient catalytic reactions that have been well adapted and evolved in a particular sequence of amino acids to provide desirable biologically relevant products with an optimum yield. While nature has understood the mechanisms and designed strategies for enzymes sequencing and function, it still remains an enigma for researchers and technologists towards a thorough understanding and fabrication of enzymes and enzymatic processes.

Here we look at two different sequences of amino acids having completely different folds and  yet catalyzing the same reaction. Typified as isoenzymes, DHFR-Ecoli chromosomal and DHFR-R67 plasmid encoded belong to different gene types (dhfrA and dhfrB), having no structural homology or sequence, catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as cofactor. While DHFR-Ecoli, a Rossman fold is an extensively  studied  enzyme and drugs have been developed to inhibit enzyme activity, one such drug trimethoprin  has been shown to be ineffective against dhfrb gene encoded SH3- domain  homotetramer R67. R67 DHFR is a homotetramer and shows numerous characteristics of a primitive enzyme including promiscuity in binding of substrate/cofactor, formation of non-productive complexes, and the absence of a conserved acid in its active-site. Furthermore, R67's active-site is a pore, which is mostly accessible by bulk solvent. This present study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one half of the active site pore using numerous interactions with R67. Also stacking between the nicotinamide ring of cofactor and the pteridine ring of substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the para-aminobenzoylglutamate tail of DHF occur in the other half of the pore due to ion-pair switching between symmetry related K32 residues from two subunits. The tail movement at the edge of the active-site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes transition state formation. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF tail angle, accomplished by different elements of the dissimilar protein folds.