title: Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
abstract: Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence 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 activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, Montreal Joint Centre for Structural Biology, Montreal, Quebec H4P 2R2 Canada.
authors.forename: A
authors.initials: A
authors.lastname: Schmidt
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: R E
authors.initials: RE
authors.lastname: MacKenzie
authors.forename: V J
authors.initials: VJ
authors.lastname: Chen
authors.forename: J R
authors.initials: JR
authors.lastname: Bewly
authors.forename: J E
authors.initials: JE
authors.lastname: Ray
authors.forename: J E
authors.initials: JE
authors.lastname: Toth
authors.forename: M
authors.initials: M
authors.lastname: Cygler
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004330:Drosophila; D004926:Escherichia coli / Q000201:enzymology; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013763:Tetrahydrofolates / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; C055342:methylene tetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases