A Functional Role for Glutamine 183 in the Folate Half‐Reaction of Methylenetetrahydrofolate Reductase from E. coli

Abstract

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes a ping‐pong reaction with NADH and 5,10‐methylenetetrahydrofolate (CH2‐H4folate) as substrates. In humans, mutations in MTHFR have been correlated with elevated levels of homocysteine, a risk factor for cardiovascular disease, and with neural‐tube defects in the fetus. We study MTHFR from Escherichia coli as a model for the catalytic domain of the human enzyme. This work focuses on Glutamine 183, an invariant, active site amino acid. X‐ray crystal structures [Biochemistry (2005) 44(34), 11447–11457] indicate that Gln 183 makes key hydrogen‐bonding interactions with both substrates. We have created the variants Gln183Glu and Gln183Ala and have begun characterization of the enzymes by steady‐state and stopped‐flow kinetics and by measurement of the flavin redox potentials. Our data suggest a major role for Gln 183 in folate binding and catalysis. Compared to the wild‐type enzyme, the Gln183Ala variant binds CH2‐H4folate with 20‐fold decreased affinity and catalyzes the folate half‐reaction at a 12‐fold decreased rate. The Gln183Glu mutant appears less impaired in the folate half‐reaction than the Gln183Ala variant. NADH binding and catalysis have not been altered by either mutation. Research supported by ACS‐PRF grant #39599‐GB4, HHMI grants #71100‐503702 and #52006298 to Grinnell College.