The 3-D Structure of a folate-dependent dehydrogenase / cyclohydrolase bifunctional enzyme at 1.5A resolution
The interconversion of two major folate one-carbon donors occurs through the sequential activities of AND(P)-dependent methylene[H{sub 4}]folate dehydrogenase (D) and methenyl[H{sub 4}]folate cyclohydrolase (C). These activities often coexist as part of a multifunctional enzyme and there are several lines of evidence suggesting that their substrates bind at overlapping sites. Little is known, however, about the nature of this site or the identity of the active-site residues for this enzyme family. We have determined, to 1.5 {angstrom} resolution, the structure of a dimer of the D/C domain of the human trifunctional 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 residues and NADP is bound along one wall. The NADP-binding domain has a Rossmann fold, characterized by a modified diphosphate-binding loop fingerprint - GXSXXXG. Dimerization occurs by antiparallel interaction of two NADP-binding domains. Superposition of the two subunits indicates domain motion occurs about a well-defined hinge region. Analysis of the structure suggests strongly that folate-binding sites for both activities are within the cleft, providing direct support for the proposed overlapping site model. The orientation of the nicotinamide ring suggests that in the dehydrogenase-catalyzed reaction hydride 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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 930066
- Report Number(s):
- BNL-80693-2008-JA; TRN: US200822%%1289
- Journal Information:
- Structure, Vol. 6
- Country of Publication:
- United States
- Language:
- English
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