PTERIN AND FOLATE REDUCTION BY THE LEISHMANIA-TARENTOLAE H-LOCUS SHORT-CHAIN DEHYDROGENASE REDUCTASE PTR1/

Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in t he protozoan parasite Leishmania. Genetic analysis has previously impl icated PTR1 in pterin and folate metabolism, PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits N ADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities, The highest activity was found with the most oxi dized pterins. The active protein was found to be a tetramer as demons trated by gel-filtration chromatography, Kinetic constants (K-m), as d etermined by double-reciprocal plots, were calculated for NADPH and fo r several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K-m of 16.9 mu M for the cofactor NADPH and K, values ranging from 3 .5 to 85 mu M for the various substrates. The dissociation constant (K D), as determined by fluorescence titration, for NADPH was estimated t o be 130 mu M. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania sh ould be useful for structure-function analysis and for developing spec ific inhibitors against this putative important chemotherapeutic targe t. (C) 1997 Academic Press.