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PubMed1996

Site-directed mutagenesis of putative catalytic and nucleotide binding sites in N10-formyltetrahydrofolate synthetase.

To determine the importance of specific amino-acid residues in catalysis and substrate binding by N10-formylH4 folate synthetase, one lysine and three histidine residues in the enzyme from Clostridium cylindrosporum were mutated to glutamine and serine residues, respectively. These residues, Lys-71, His-125, His-131, and His-268, are conserved in four bacterial and five eukaryotic proteins for which the amino-acid sequences are known. Previous evidence indicated that a histidine residue may play a role in catalysis and it has been proposed that Lys-71 could be a member of a putative nucleotide binding consenus sequence. The histidine mutations, H125S, H131S, and H268S, produced proteins that were unstable and were proteolytically degraded to different extents. No activity of purified H268S could be detected and the 240 kDa native tetramer was also absent. Activities of the H125S and H131S mutants could be measured and the Km values of the substrates were similar to those for the wild-type enzyme. It is concluded that the mutations resulted in monomers that do not fold properly and/or do not associate to the active tetramer and, as a consequence, are susceptible to intracellular proteolytic digestion. On the other hand, the K71Q mutation did not produce proteolyzed material. The resulting protein had a kcat value which was reduced by a factor of 3.3 x 10(-4). Km values of the substrates were not affected, nor were the affinty constants for MgATP and H4PteG3. CD and fluorescence spectra demonstrated that little change in the tertiary structure of the protein had occurred as a result of the mutation. The monomer form of K71Q was less stable than the monomer of the wild-type enzyme and reassociated less efficiently than the wild-type. From these results it is suggested that Lys-71 plays a critical role in catalysis by N10-formylH4 folate synthetase and that this residue may reside at an intersubunit interface.

Amino Acid Sequence↗
PubMed1991

A pseudogene on the X chromosome for the human trifunctional enzyme MTHFD (methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase).

The human trifunctional folate-dependent protein MTHFD has been mapped to chromosome 14q24 and to the X chromosome was identified by screening an X-chromosome-specific library. Amplification by the polymerase chain reaction (PCR) and sequencing of PCR products indicate that the sequence is an intronless pseudogene.

Aminohydrolases↗
PubMed1994

Conformation of ATP and ADP bound to N10-formyltetrahydrofolate synthetase determined by TRNOE NMR spectroscopy.

ATP and ADP bind to N10-CHO-H4folate synthetase from Clostridium cylindrosporum at four identical sites. Although both ADP and ATP bind to the enzyme with essentially the same Ka values as the Mg2+.nucleotide complexes, only the Mg2+.nucleotides are kinetically active. Using transferred nuclear Overhauser effect (TRNOE) NMR spectroscopy, we have measured the time-dependent NOE buildup rates of selected protons in ADP and ATP bound to N10-CHO-H4folate synthetase after preirradiating protons H1', H2', H3', and H4'. The results were used to calculate interproton distances. In order to define the conformations of ADP and ATP bound to the enzyme, we used the TRNOE distance constraints in a distance geometry algorithm. The results of the distance geometry calculations suggest that, within experimental error, the conformations of both ADP and ATP (with or without Mg2+) have an average glycosidic torsion angle X (O4'-C1'-N9-C8) of 100 degrees +/- 20 degrees and a sugar pucker angle psi' (C5'-C4'-C3'-O3') of 85 degrees +/- 5 degrees. These values are consistent with a nucleotide structure generated by computer modeling after energy minimization, which has X = 90 degrees +/- 6 degrees and psi' = 81 degrees, indicating a high-anti and C3'-endo conformation.

Adenosine Diphosphate↗
PubMed1996

Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4.

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P6(1) and P6(5), with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 A. The P6(1) structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Reyes, V.M., Sawaya, M.R., Brown, K.A., & Kraut, J. (1995) Biochemistry 34, 2710-2723]; enzyme and ligand conformations are very similar to the P6(1) 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P6(1) structure are nearly in the closed conformation, exemplified by the methotrexate P6(1) binary complex, in the P6(5) structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S.J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P6(1) structure, in which the Met-20 loop (residues 9-23) is turned away from the substrate binding pocket, in the P6(5) structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P6(5) structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P6(1) structure it lies at the boundary between the two molecules of the asymmetric unit, while in P6(5) it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 3(10) helix (residues 16-18 in the Met-20 loop), a second unique feature of the P6(5) crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol-keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies that site as part of a hypothetical proton-relay mechanism. Two arginine side chains, Arg-52 in the P6(5) structure and Arg-44 in molecule A of the P6(1) structure, are turned away drastically from the ligand (p-aminobenzoyl)glutamic acid moiety as compared with previously reported DHFR binary complex structures. As in the ecDHFR dideazatetrahydrofolate complex, in both the P6(1) and P6(5) structures a water molecule bridges pteridine O4 and Trp-22(N epsilon 1) with ideal geometry for hydrogen bonding, perhaps contributing to the slow release of 5,6,7,8-tetrahydrofolate from the enzyme-product complex. When either the P6(1) or the P6(5) structures are superimposed with the NADPH holoenzyme [Sawaya, M. R. (1994) Ph.D. Dissertation, University of California, San Diego], we find that the distances between the nicotinamide C4 and pteridine C6 and C7 are very short, 2.1 and 1.7 A in the P6(1) case and 2.0 and 1.4 A in the P6(5) case, perhaps in part explaining the more rapid release of tetrahydrofolate from the enzyme-product complex when NADPH is bound.

4-Aminobenzoic Acid↗
PubMed1998

Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes.

The kinetic properties of three methylenetetrahydrofolate dehydrogenase-cyclohydrolase (D/C) enzymes (the NADP-dependent bifunctional domain of the human cytoplasmic trifunctional enzyme, the human mitochondrial NAD-dependent bifunctional enzyme, and the NAD(P)-dependent bifunctional enzyme from Photobacterium phosphoreum) were determined in both forward and reverse directions. In the forward direction, the enzymes possess widely different ratios of kcat C/Kcat D, but all channel methenylH4folate produced by the D activity to the C activity with approximately the same efficiency. A deuterium isotope effect is observed with the human NADP-dependent enzyme in both forward and reverse dehydrogenase assays, consistent with hydride transfer being rate limiting for the interconversion of methenyl- and methyleneH4folate. However, no kinetic isotope effect is observed for the overall reverse reaction (formylH4folate to methyleneH4folate). We devised an assay to measure the reverse cyclohydrolase activity independent of the dehydrogenase, and determined that the Kcat (overall reverse) for each enzyme is approximately equal to the Kcat for its reverse cyclohydrolase activity. Therefore, the rate-limiting step in the overall reverse reaction is not hydride transfer by the dehydrogenase, but the production of methenylH4folate catalyzed by the cyclohydrolase. The reverse cyclohydrolase activities of the NADP-dependent D/C and the P. phosphoreum enzymes, but not the mitochondrial NAD-dependent enzyme, can be stimulated 2-fold by the addition of 2',5'-ADP. The results suggest that the cyclohydrolases of the human NADP dependent and P. phosphoreum enzymes are optimized to catalyze the reverse reaction in the presence of bound coenzyme. These results imply that essentially all of the methenylH4folate produced by the cyclohydrolase in the reverse reaction is channeled to the dehydrogenase.

Adenosine Diphosphate↗
PubMed2001

Bioactivity of [6R]-5-formyltetrahydrofolate, an unusual isomer, in humans and Enterococcus hirae, and cytochrome c oxidation of 10-formytetrahydrofolate to 10-formyldihydrofolate.

The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H(4)F), is not found in Nature. An oral dose of 13.5 micromol of [6R]-5-HCO-H(4)F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H(4)F) with second-order kinetics and a rate constant of 1.3 x 10(4) M(-1) x s(-1). The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H(2)F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H(4)F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H(4)F from 5-HCO-H(4)F, the unusually large rate constant for the oxidation of 10-HCO-H(4)F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H(4)F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H(4)F to 10-HCO-H(2)F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer.

Cytochrome c Group↗
PubMed1995

Mechanism for the coupling of ATP hydrolysis to the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate.

5,10-Methenyltetrahydrofolate synthetase catalyzes the irreversible conversion of 5-formyl-tetrahydropteroylpolyglutamates (5-CHO-H4PteGlu(n)) to 5,10-methenyltetrahydropteroylpolyglutamates (5, 10-CH(+)-H4PteGlu(n)). The equilibrium of the nonenzymatic reaction, which equilibrates slowly in the absence of enzyme, greatly favors 5-CHO-H4PteGlu(n). The enzyme couples the reaction to the hydrolysis of ATP shifting the equilibrium to favor 5,10-CH(+)-H4PteGlu(n). Substrate-dependent non-equilibrium isotope exchange of [3H]ADP into ATP was observed, suggesting the formation of a phosphorylated intermediate of 5-CHO-H4PteGlu(n) during the enzyme-catalyzed reaction. The competitive inhibitor 5-formyltetrahydrohomofolate also supported the ADP to ATP exchange, suggesting that this molecule could also form a phosphorylated intermediate. The initial rates of the ADP-ATP exchange with saturating ADP were about 70 s-1 for both compounds, while the kcat values for product formation were 5 s-1 for 5-CHO-H4PteGlu(n) and 0.005 s-1 for 5-formyltetrahydrohomofolate. Starting with 5(-)[18O]CHO-H4PteGlu(n), it was shown by 31P NMR that the formyl oxygen of the substrate was transferred to the product phosphate during the reaction. This further supports the existence of a phosphorylated intermediate. The formyl group of 5-CHO-H4PteGlu(n) is known to be an equilibrium mixture of two rotamers. Stopped-flow analysis of the enzymatic reaction showed that only one of the rotamers serves as a substrate for the enzyme.

Adenosine Triphosphate↗