Modular organization of FDH: Exploring the basis of hydrolase catalysis

Domain organization is an intrinsic element of protein structure. The majority of proteins consist of distinctive domains that can act independently or cooperatively to achieve a unique function (Vogel et al. 2004; Bornberg-Bauer et al. 2005). The interaction between functional modules within a single polypeptide determines mechanisms by which multidomain proteins operate. An interesting example of a multidomain protein is the chimeric folate enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH). FDH consists of two distinct catalytic domains, the amino-terminal and carboxy-terminal, connected by an intermediate linker (Fig. ​(Fig.1)1) (Cook et al. 1991; Krupenko et al. 1997a). The carboxy-terminal domain of FDH (Ct-FDH), an aldehyde dehydrogenase (ALDH) homolog (Cook et al. 1991), is capable of catalyzing an NADP+-dependent aldehyde dehydrogenase reaction utilizing short-chain aldehydes as a substrate (Fig. ​(Fig.1)1) (Krupenko et al. 1997b). The amino-terminal domain (Nt-FDH) is a homolog of the formyl transferase enzymes L-methionyl-tRNA formyltransferase (FMT) and glycinamide ribonucleotide formyltransferase (GARFT) (Cook et al. 1991). Similar to FDH, these enzymes utilize 10-formyltetrahydrofolate (10-formyl-THF) as a substrate. Nt-FDH, however, does not possess transferase activity but instead hydrolyzes 10-formyl-THF to yield formate and THF (Fig. ​(Fig.1)1) (Cook and Wagner 1995). The intermediate domain of FDH does not have significant homology with any known protein, nor does it have any known catalytic activity (Cook et al. 1991). 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 ... The two catalytic domains of FDH, connected by an intermediate linker, work in concert to create a new enzymatic activity: The full-length FDH converts 10-formyl-THF to tetrahydrofolate (THF) in an NADP+-dependent dehydrogenase reaction in which the formyl group is oxidized to CO2 (Min et al. 1988). Of note, within the full-length FDH, both catalytic domains are still capable of independently catalyzing their intrinsic reactions (Cook et al. 1991). It is not known, however, whether these reactions have any physiological significance. On the other hand, the main FDH activity, which requires the concerted action of both catalytic domains, can have a strong impact on the cell. Recent studies from this laboratory have shown that expression of FDH in FDH-deficient cancer cells inhibits proliferation, leading to apoptosis (Krupenko and Oleinik 2002; Oleinik and Krupenko 2003). Importantly, these effects can be produced only by catalytically functional, full-length FDH (Krupenko and Oleinik 2002). Therefore, understanding how the two domains act together to create a new enzymatic mechanism is crucial for understanding the biological role of FDH. The shuffling of pre-existing functional modules is considered to be a common mechanism of protein evolution (Patthy 2003), and using domain replacement to produce functional hybrids has become an important approach for studying multidomain proteins in recent years (Bhatt et al. 2004; Sasata et al. 2004; Fan et al. 2005; Lu et al. 2005; Suo 2005). Such studies have been done for the Nt-FDH homologous enzyme, GARFT (Nixon et al. 1997; Nixon and Benkovic 2000; Lee et al. 2003). GARFT is ~200 amino acid residues long and consists of two domains, a folate-binding amino-terminal domain and a GAR-binding carboxy-terminal domain (Greasley et al. 1999). Domain replacement studies have demonstrated that functional hybrids containing GARFT activity can be produced from the GAR-binding domain and the folate-binding domain originated from other 10-formyl-THF utilizing enzymes (Nixon et al. 1997; Nixon and Benkovic 2000; Lee et al. 2003). This implies that individual modules (which can be considered subdomains) of GARFT have at least some functional independence. The recently solved crystal structure of Nt-FDH revealed that its overall fold and architecture of the catalytic center are remarkably similar to those of FMT or GARFT (Chumanevich et al. 2004). Specifically, the two catalytic residues whose importance has been established for all three enzymes (Inglese et al. 1990; Warren et al. 1996; Krupenko and Wagner 1999; Newton and Mangroo 1999; Shim and Benkovic 1999; Krupenko et al. 2001), aspartate and histidine, overlay very closely in these proteins (Chumanevich et al. 2004). This suggests a similar catalytic mechanism for removing the formyl group from the folate substrate. Nt-FDH, however, is missing an asparagine in position 104, a crucial residue in FMT and GARFT catalysis (Newton and Mangroo 1999; Shim and Benkovic 1999), and instead contains an isoleucine at this position. When compared to GARFT, Nt-FDH and FMT are extended at the carboxyl terminus by ~100 residues (Krupenko and Wagner 1998). This part of their respective sequences represents a separate domain, which in the case of FMT binds methionyl-tRNA (Schmitt et al. 1996). In spite of low sequence identity, these carboxy-terminal regions of FMT and Nt-FDH have surprisingly similar structures (Chumanevich et al. 2004). There is no known function for this region in Nt-FDH, however. We have undertaken the present study to understand the modular nature of FDH through the generation of hybrid enzymes by domain replacement with FMT, and to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes.