Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase
A. Schmidt et al., Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase, BIOCHEM, 39(21), 2000, pp. 6325-6335
Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceu
tical interest, as their function is crucial for amino acid and DNA biosynt
hesis. The crystal structure of the human cytosolic methylenetetrahydrofola
te dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme ha
s been determined previously with a bound NADP cofactor. While the substrat
e binding site was identified to be localized in a deep and rather hydropho
bic cleft at the interface between two protein domains, the unambiguous ass
ignment of catalytic residues was not possible. We succeeded in determining
the crystal structures of three ternary DC301/NADP/inhibitor complexes. In
vestigation of these structures followed by site-directed mutagenesis studi
es allowed identification of the amino acids involved in catalysis by both
enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues o
f a strictly conserved motif for active sites in dehydrogenases. While Lys5
6 is in a good position for chemical interaction with the substrate analogu
e, Tyr52 was found stacking against the inhibitors' aromatic rings and henc
e seems to be more important for proper positioning of the ligand than for
catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activ
ator for water in the cyclohydrolase step. These and the other residues (Gl
n100 and Asp125), with which contacts are made, are strictly conserved in T
HF dehydrogenases. On the basis of structural and mutagenesis data, we prop
ose a reaction mechanism for both activities, the dehydrogenase and the cyc
lohydrolase.