THE 3-D STRUCTURE OF A FOLATE-DEPENDENT DEHYDROGENASE CYCLOHYDROLASE BIFUNCTIONAL ENZYME AT 1.5 ANGSTROM RESOLUTION/

Background: The interconversion of two major folate one-carbon donors occurs through the sequential activities of NAD(P)-dependent methylene [H-4]folate dehydrogenase (D) and methenyl[H-4]folate cyclohydrolase ( C). These activities often coexist as part of a multifunctional enzyme and there are several lines of evidence suggesting that their substra tes bind at overlapping sites. Little is known, however, about the nat ure of this site or the identity of the active-site residues for this enzyme family, Results: We have determined, to 1.5 Angstrom resolution , the structure of a dimer of the D/C domain of the human trifunctiona l cytosolic enzyme with bound NADP cofactor, using the MAD technique, The D/C subunit is composed of two alpha/beta domains that assemble to form a wide cleft. The cleft walls are lined with highly conserved re sidues and NADP is bound along one wall, The NADP-binding domain has a Rossmann fold, characterized by a modified diphosphate-binding loop f ingerprint - GXSXXXG. Dimerization occurs by antiparallel interaction of two NADP-binding domains, Superposition of the two subunits indicat es domain motion occurs about a well-defined hinge region. Conclusions : Analysis of the structure suggests strongly that folate-binding site s for both activities are within the cleft, providing direct support f or the proposed overlapping site model, The orientation of the nicotin amide ring suggests that in the dehydrogenase-catalyzed reaction hydri de transfer occurs to the pro-R side of the ring, The identity of the cyclohydrolase active site is not obvious, We propose that a conserved motif - Tyr52-X-X-X-Lys56 - and/or a Ser49-Gln100-Pro102 triplet have a role in this activity.