Modular organization of FDH: Exploring the basis of hydrolase catalysis

Abstract

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.

Figure 1.

(A) Reactions catalyzed by FDH (the full-length FDH catalyzes all three reactions; the amino-terminal domain catalyzes the hydrolase reaction; the carboxy-terminal domain catalyzes the aldehyde dehydrogenase reaction). (B) Schematic diagram of FDH monomer (blue, the amino-terminal domain; gray, the carboxy-terminal domain; black, the intermediate linker) and FMT/FDH hybrid proteins, showing portions of N_t-FDH (blue) and FMT (green) within each hybrid. (C) Partial sequence alignments of FDH, FMT, and hybrid proteins showing the amino-terminal splice junction (top) and the carboxy-terminal splice junction (bottom). Dark-shaded residues are identical, whereas light-shaded residues are similar. Residues originating from FDH are shown in blue and those from FMT in green. Unique residues created by the insertion of restriction sites are in red. Note that hybrid III has a carboxy-terminal splice junction identical to that of hybrid I. The alignment was prepared using MacVector. (D) Ribbon presentation of crystal structures of FMT (upper left) (PDB ID 2FMT) and N_t-FDH (upper right) (PDB ID 1S3I) along with models of the N_t-FDH homologous region of hybrid II (lower left) and hybrid III (lower right). Hybrid models were prepared using SYBYL 7.0. FMT and N_t-FDH structures were superimposed, and relevant portions of each protein were merged together in a separate area. Hybrid structures were generated by creating bonds between the chains. The resulting hybrid structures were energy-minimized. Visual depictions were generated using MOLSCRIPT (Kraulis 1991).

Figure 2.

Activity of hybrid III as compared to the wild-type FDH. Error bars represent standard error of at least three measurements.

Figure 3.

(A) Spatial arrangement of essential catalytic residues and folate substrate within the N_t-FDH active site. This figure was generated by superimposing the structure of N_t-FDH (PDB ID 1S3I) with that of GARFT complexed with the inhibitor 10-formyl-5,8,10-trideazafolate (PDB ID 1C2T). Superimposed isoleucine (wild-type enzyme) and asparagine (I104N mutant) are shown at position 104. (B) Backbone of N_t-FDH showing the SLLP motif. Hydrogen bonds are shown as dashed lines.

Figure 4.

Activities of full-length FDH mutants expressed as a percentage of wild type. Error bars represent standard error of at least three measurements.