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.
10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N-and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP ؉ -dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO 2 , consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP ؉ -dependent oxidation of the formyl group to CO 2 in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protein. The subunit of 10-formyltetrahydrofolate dehydrogenase (FDH 3 ; ALDH1L1; EC 1.5.1.6) represents a single polypeptide consisting of three domains, with two of these domains possessing their own catalytic activities (1-4). The N-terminal domain of FDH (residues 1-310) functions as 10-formyltetrahydrofolate (10-fTHF) hydrolase, converting 10-fTHF to tetrahydrofolate (THF) and formate ( Fig. 1) (2). The C-terminal domain (residues 420 -902) is an aldehyde dehydrogenase-homologous enzyme capable of NADP ϩ -dependent oxidation of short chain aldehydes to the corresponding acids (3). The two catalytic domains are separated by a short (ϳ100 residues) linker domain (intermediate domain). Although the physiological significance of the catalytic functions of the separate domains of FDH is not clear, the well established function of FDH is the conversion of 10-fTHF to THF in an NADP ϩ -dependent dehydrogenase reaction (5-7). It is believed that this reaction regulates intracellular 10-fTHF/THF pools (8), controls de novo purine biosynthesis (9, 10), and affects the methylation potential of the cell (11). This reaction is observed only when the two catalytic domains are linked in one polypeptide by the intermediate domain (2). Studies of FDH structure and catalytic mechanism (12-18) suggest that this reaction proceeds in two steps: (i) hydrolytic removal of the formyl group from 10-fTHF and (ii) NADP ϩ -dependent oxidation of formyl to CO 2 . The first step takes place in the N-terminal hydrolase domain, whereas the second step occurs in the C-terminal aldehyde dehydrogenase domain (2,3,17,18).The connection between the two steps is the transfer of the formyl group between the two catalytic domains. However, the nature of such a transfer is not obvious. We have demonstrated previously that the two catalytic domains of FDH do not form close contacts in the absence of the intermediate domain (2). It has been suggested that the intermediate domain keeps the N-and C-terminal domains of FDH in close proximity to each other and in the correct orientation to create an interface between the two domains and to allow the formyl group transfer between the two catalytic centers. In support of this, t...
Of the two Ca(II) in carp muscle calcium binding parvalbumin B, one may be removed by dialysis against EGTA without significant alteration of the circular dichroism spectra at 224 nm, a result suggesting little or no change in the helical content of 47%. Binding of the two Ca(II) is not cooperative. Addition of a 20-fold or greater excess of EGTA at pH 8.5 results in removal of both Ca(II), reduces the helical content to about 39%, alters only served in Tris buffer at pH 8.5 or bis-tris buffer at pH 6.5.1 Abbreviations used are: Nbs2, 5,5'-dithiobis(2-nitrobenzoic acid); EGTA, ethylene glycol bis(/3-aminoethyl ether)-7V,yV'-tetraacetate. BIOCHEMISTRY,VOL.
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