FDH: an aldehyde dehydrogenase fusion enzyme in folate metabolism

FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO(2) in a NADP(+)-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1-310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400-902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to classes 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311-399), which is a structural and functional homolog of carrier proteins possessing a 4'-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO(2).     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis

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. A 4'-phosphopantetheine swinging arm bound through a phosphoester bond to Ser(354) of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protein and provides the previously lacking component of the FDH catalytic mechanism.     

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.     

Modular organization of FDH: Exploring the basis of hydrolase catalysis

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.     

The crystal structure of the hydrolase domain of 10-formyltetrahydrofolate dehydrogenase: mechanism of hydrolysis and its interplay with the dehydrogenase domain

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate, a precursor for nucleotide biosynthesis, to tetrahydrofolate. The protein comprises two functional domains: a hydrolase domain that removes a formyl group from 10-formyltetrahydrofolate and a NADP(+)-dependent dehydrogenase domain that reduces the formyl to carbon dioxide. As a first step toward deciphering the catalytic mechanism of the enzyme, we have determined the crystal structure of the hydrolase domain of FDH from rat, solved to 2.3-A resolution. The structure comprises two domains. As expected, domain 1 shares the same Rossmann fold as the related enzymes, methionyl-tRNA-formyltransferase and glycinamide ribonucleotide formyltransferase, but, unexpectedly, the structural similarity between the amino-terminal domain of 10-formyltetrahydrofolate dehydrogenase and methionyl-tRNA-formyltransferase extends to the C terminus of both proteins. The active site contains a molecule of beta-mercaptoethanol that is positioned between His-106 and Asp-142 and that appears to mimic the formate product. We propose a catalytic mechanism for the hydrolase reaction in which Asp-142 polarizes the catalytic water molecule and His-106 orients the carbonyl group of formyl. The structure also provides clues as to how, in the native enzyme, the hydrolase domain transfers its product to the dehydrogenase domain.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Enzymatic properties of ALDH1L2, a mitochondrial 10-formyltetrahydrofolate dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4'-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.     

Crystal structures of the carboxyl terminal domain of rat 10-formyltetrahydrofolate dehydrogenase: implications for the catalytic mechanism of aldehyde dehydrogenases

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.     

Chemical studies of high-Km aldehyde dehydrogenase from rat liver mitochondria

Studies of pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde dehydrogenase from rat liver mitochondria. Sensitized photooxidation completely arrests the bifunctional activities of the dehydrogenase. Carboxamidomethylation abolishes the dehydrogenase activity, whereas acetimidination eliminates the esterase activity. These results suggest that histidine (pKa near 6) and cysteine (pKa near 10) are likely the catalytic residues for the dehydrogenase activity, while the esterase activity is functionally related to histidine (pKa near 7) and a residue with the pKa value of 10-11. The two residues, a carboxyl group and an arginine, that discriminate between NAD+ and NADP+ are present at the coenzyme binding site of the mitochondrial high-Km aldehyde dehydrogenase from rat liver.     

Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme

The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.     

Resolution of rat liver 10-formyltetrahydrofolate dehydrogenase/hydrolase activities

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Previous studies of 10-formyltetrahydrofolate dehydrogenase purified from rat or pig liver homogenized in phosphate buffers indicated the presence of copurifying 10-formyltetrahydrofolate hydrolase activity, which catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate and formate. We find that the supernatant from rat liver homogenized in mannitol/sucrose/EDTA medium contains essentially all of the total cellular 10-formyltetrahydrofolate dehydrogenase activity, but no measurable hydrolase activity. Treating mannitol/sucrose/EDTA-washed mitochondria with Triton X-100 (0.5%) releases hydrolase activity in soluble form. 10-Formyltetrahydrofolate dehydrogenase purified from the mannitol/sucrose/EDTA supernatant has no 10-formyltetrahydrofolate hydrolase activity. Results of kinetic experiments using the hydrolase-free dehydrogenase give a complex rate equation with respect to (6R,S)-10-formyltetrahydrofolate. Double-reciprocal plots fit a 2/1 hyperbolic function with apparent Km values of 3.9 and 68 microM. Our results indicate that 10-formyltetrahydrofolate hydrolase and dehydrogenase are not alternate catalytic activities of a single protein, but represent two closely related and separately compartmentalized hepatic enzymes.     

Acyl Carrier Protein-specific 4��-Phosphopantetheinyl Transferase Activates 10-Formyltetrahydrofolate Dehydrogenase

4′-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4′-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO₂. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G₁ arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.     

Knocking down 10-formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement

Background

Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO₂, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase.

Methods

10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC.

Results

The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain.

Conclusions

10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis.

General significance

These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.     

Rat liver 10-formyltetrahydrofolate dehydrogenase, carbamoyl phosphate synthetase 1 and betaine homocysteine S-methytransferase were co-purified on Kunitz-type soybean trypsin inhibitor-coupled sepharose CL-4B

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.     

Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre

Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.     

ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase

Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO₂ in an NADP⁺-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO₂ production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.     

NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. III. Comparative analysis

The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.     

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.     

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.