Crystal Structures of the Carboxyl Terminal Domain of Rat 10-Formyltetrahydrofolate Dehydrogenase:  Implications for the Catalytic Mechanism of Aldehyde Dehydrogenases

Abstract

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.

Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

“…On this basis, stabilization of a free thiolate in direct contact with the NAD+ cofactor should not be possible. Re-examination of the crystal structures of two distinct members of the ALDH superfamily, formyltetrahydrofolate dehydrogenase and mitochondrial ALDH2 (Cα rmsd 1.36Å), now confirms that this direct interaction actually leads to a cysteine-cofactor adduct, one which is further confirmed by a broad absorption maximum around 319nm (9). The adduct bond would have to break before the catalytic cycle could proceed.…”

“…Prior to our presentation on the adduct in July 2006 (22), this reaction had only been reported by adding the respective reactants (thiols plus nicotinamides) together in solution (8). We surmised that another configuration of atoms was probably responsible for describing the chemistry of ALDH until recent crystallographic data (9) confirmed that this simulation result was correct in that this adduct is not only observed in the crystal structure of formyltetrahydrofolate dehydrogenase but also in the ALDH2 structures from re-examination of the earlier data of Perez-Miller and Hurley (1). The mechanistic implications of this adduct are many-fold.…”

“…Thus, from this particular arrangement of atoms taken from the crystal structure, formation of the adduct is much more favorable than formation of the oxyanion intermediate even given the errors associated with the PM3 method. Experiments (9) show degradation of the adduct by soaking crystals in aldehydes but provide no information as to the thermodynamics of each individual reaction. Although formation of the adduct must surely hinder the efficiency of catalysis in ALDH, the equilibrium between Cys302 with NAD + in the HY position (reactants separated) and the adduct is heavily weighted toward the separated reactants (9).…”

Conserved Catalytic Residues of the ALDH1L1 Aldehyde Dehydrogenase Domain Control Binding and Discharging of the Coenzyme

“…The resulting thiolate ion is likely stabilized by the positively charged nic-otinamide ring of the coenzyme and/or adjacent main chain amide groups. It has also been suggested that, in tetrameric class 1 and 2 ALDHs, the proton abstracted by Glu-268 is shuttled to bulk water (10,25). In addition, Glu-268 is also the most likely residue activating a water molecule in the deacylation step of the reaction (5,7,10,23,26).…”

“…A distinct feature of ALDHs is the apparent requirement for coenzyme flexibility in the process of catalysis (10,(27)(28)(29). For hydride transfer to occur, in the first step of this reaction the C4 atom of the nicotinamide ring has to be in close proximity to the catalytic cysteine.…”