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PubMed1992

Human dihydrofolate reductase: reduction of alternative substrates, pH effects, and inhibition by deazafolates.

The kinetics of the NADPH-dependent reduction of 7,8-dihydrofolate, folate, and 7,8-dihydrobiopterin by human dihydrofolate reductase have been examined over the pH range from 4.0 to 9.5. The V and V/K profiles obtained with the three substrates indicate that a single ionizing residue at the active site of the enzyme must be protonated for catalysis. Both the maximum velocity of the reactions and the rate of interaction of the substrates with the enzyme-NADPH complex decrease in the order dihydrofolate greater than dihydrobiopterin much greater than folate. From the pK values of the V/K profiles, it can be concluded that, while dihydrofolate behaves as a sticky substrate and dihydrobiopterin exhibits slight stickiness, folate is not a sticky substrate. Further support for this conclusion comes from the results of deuterium isotope effects. The pK values obtained from both the V and V/Kfolate profiles are similar to the intrinsic pK value of 5.6 for both the free enzyme and the enzyme-NADPH complex. The folate analogue, 5-deazafolate, is not a substrate, but it undergoes strong interaction with the enzyme. This interaction, which is enhanced by the presence of NADPH, is due to protonation of the bound ligand that does not involve the single ionizing group at the active center of the enzyme. Difference spectra yield evidence for the protonation of bound 5-deazafolate and show that, on binding to the enzyme-NADPH complex, the pK of the N-8 atom is raised to about 10 from a value of about 4 in solution. The results are in accord with those of a recent paper on the three-dimensional structure of the enzyme-5-deazafolate complex [Davies, J.F., Delcamp, T.J., Prendergast, N.J., Ashfors, V.A., Freisheim, J.H., & Kraut, J. (1990) Biochemistry 29, 9467-9479] which indicate that there is hydrogen bond formation between N-8 of the ligand and the carbonyl group of Ile-7. However, the present findings do not support the idea that bound 5-deazafolate resembles the transition-state complex for folate reduction. Quinazolines also interact strongly with the enzyme but in a pH-independent manner. The dissociation constants for the binary complexes are an order of magnitude lower than that for the binding to the enzyme of unprotonated 5-deazafolate. This difference reflects the hydrophobic nature of the amino acid residues at the active site that are near the N-5 and N-8 nitrogens of bound pterins.

Folic Acid↗

PubMed2001

Interloop contacts modulate ligand cycling during catalysis by Escherichia coli dihydrofolate reductase.

As a continuation to our studies on the importance of interloop interactions in the Escherichia coli DHFR catalytic cycle, we have investigated the role of the betaG-betaH loop in modulating the closed and occluded conformations of the Met20 loop during the DHFR catalytic cycle. Specifically, to assess the importance of the hydrogen bond formed between Ser148 in the betaG-betaH loop and the Met20 loop, Ser148 was independently substituted with aspartic acid, alanine, and lysine. Moreover, the betaG-betaH loop was deleted entirely to yield the Delta(146-148) DHFR mutant. Steady-state turnover rates for all mutants were at most 3-fold lower than the wild-type rate. Lack of an isotope effect on this rate indicated the chemistry step does not contribute to the steady-state turnover. Consistent with this finding, hydride transfer rates for the DHFR mutants were at least 10-fold greater than the observed steady-state rates. The values ranged from a 30% decrease (Ser148Ala and Ser148Lys) to a 50% increase (Ser148Asp) in rate relative to that of the wild type. Modifications of the betaG-betaH loop enhanced the affinity for the cofactor and decreased the affinity for pterin, as determined by the K(D) values of the mutant proteins. Further analysis of Ser148Ala and Delta(146-148) DHFRs indicated these effects were manifest mainly in ligand off rates, although in some cases the on rate was affected. The Ser148Asp and Delta(146-148) mutations perturbed the preferred catalytic cycle through the introduction of branching at key intermediates. Rather than following the single WT pathway which involves loss of NADP(+) and rebinding of NADPH to precede loss of the product H4F (negative cooperativity), the mutants can reenter the catalytic cycle through different pathways. These findings suggest that the role of the interloop interaction between the betaG-betaH loop and the Met20 loop is to modulate ligand off rates allowing for proper cycling through the preferred kinetic pathway.

Catalysis↗

PubMed1994

Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

Binding Sites↗

PubMed1993

An examination of the copper requirement of phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared "copper-free" CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The "copper-free" enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the "copper-free" preparation, indicating that the active hydroxylating species may not require a redox-active metal. The KdS for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 microM, respectively. Both copper and zinc were found to be potent inhibitors of "copper-free" CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed2001

Determination of an optimal potential window for catalysis by E. coli dimethyl sulfoxide reductase and hypothesis on the role of Mo(V) in the reaction pathway.

Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential. The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)). At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0). Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance. The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state.

Binding Sites↗

PubMed2001

Interligand Overhauser effects in type II dihydrofolate reductase.

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to the increased clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The type II enzymes contain four identical subunits which form a homotetramer containing a single active site pore accessible from either end. Although the crystal structure of the complex of R67 DHFR with folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], the nature of the ternary complex which must form with substrate and cofactor is unclear. We have performed transferred NOE and interligand NOE (ILOE) studies to analyze the ternary complexes formed from NADP(+) and folate in order to probe the structure of the ternary complex. Consistent with previous studies of the binary complex formed from another type II DHFR, the ribonicotinamide bond of NADP(+) was found to adopt a syn conformation, while the adenosine moiety adopts an anti conformation. Large ILOE peaks connecting NADP(+) H4 and H5 with folate H9 protons are observed, while the absence of a large ILOE connecting NADP(+) H4 and H5 with folate H7 indicates that the relative orientation of the two ligands differs significantly from the orientation in the chromosomal enzyme. To obtain more detailed insight, we prepared and studied the folate analogue 2-deamino-2-methyl-5,8-dideazafolate (DMDDF) which contains additional protons in order to provide additional NOEs. For this analogue, the exchange characteristics of the corresponding ternary complex were considerably poorer, and it was necessary to utilize higher enzyme concentrations and higher temperature in order to obtain ILOE information. The results support a structure in which the NADP(+) and folate/DMDDF molecules extend in opposite directions parallel to the long axis of the pore, with the nicotinamide and pterin ring systems approximately stacked at the center. Such a structure leads to a ternary complex which is in many respects similar to the gas-phase theoretical calculations of the dihydrofolate-NADPH transition state by Andres et al. [(1996) Bioorg. Chem. 24, 10-18]. Analogous NMR studies performed on folate, DMDDF, and R67 DHFR indicate formation of a ternary complex in which two symmetry-related binding sites are occupied by folate and DMDDF.

Binding Sites↗

PubMed1983

Purification and properties of dihydroorotate oxidase from Crithidia fasciculata and Trypanosoma brucei.

Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide. A variety of low molecular weight oxidants (e.g., quinones or ferricyanide) may replace oxygen as the electron acceptor during catalysis. The dihydroorotate oxidase of T. brucei was purified 1400-fold to apparent homogeneity by a highly similar isolation procedure. The estimated native (Mr 62 000) and subunit (Mr 30 500) molecular weights indicated a dimeric protein comparable in size to the enzyme from Crithidia. These results suggest that dihydroorotate oxidation is mediated by flavoprotein oxidases in these parasitic protozoa rather than by pterin-linked hydroxylases as recently proposed [Kidder, G. W., & Nolan, L.L. (1973) Biochem. Biophys. Res. Commun. 53, 929-936; Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Animals↗

PubMed1985

Tetrahydrobiopterin analogues: solution conformations of 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyltetrahydropterins as determined by proton nuclear magnetic resonance.

The conformation of tetrahydrobiopterin analogues in aqueous solution at 23 degrees C has been determined by analyzing the 200-MHz 1H NMR spectral parameters of the enzymatically active 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyl-5,6,7,8-tetrahydropterins. Each of these cofactors, with the exception of the cis-6,7-dimethyl isomer, exhibited an unusually small trans 6H-7H spin-spin coupling (8.5-9.1 Hz). An empirical equation that accounts for the effects of substituent electronegativity and orientation on vicinal couplings [Haasnoot, C. A. G., deLeeuw, F. A. A. M., & Altona, C. (1980) Tetrahedron 36, 2783-2792] predicted this coupling to be 11.3-11.6 Hz. We attribute the discrepancy between the calculated and experimentally observed values of this coupling to hyperconjugation of the axially oriented C7-H bond with the pi orbital of the vinylogous amide protein of the pterin ring (N8-C8a = C4a-C4 = O) rather than conformational averaging. The trans 6H-7H interproton distance in the 6-methyl analogue is calculated to be 3.0 A from the measured decrease in the spin-lattice relaxation rate of the axially oriented C7 proton after specific deuteration at C6. This is consistent with the single-conformer interpretation. Chemical shift comparisons of the methyl resonances of these analogues, NOE measurements from selectively deuterated analogues, and the differential sensitivities of axially vs. equatorially disposed ring protons to protonation at N5 all indicate that (i) the methyl substituents at both the C6 and C7 positions markedly prefer equatorial-like orientations and (ii) the tetrahydropterin ring is, with the exception of a pronounced pucker at C6, nearly planar.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopterins↗

PubMed1986

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme.

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

Chromobacterium↗

PubMed1988

Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants.

Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate). Both chromophores are fluorescent, and either can function as a sensitizer in catalysis. At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2. Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates [UV-oligo(dT)n] quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation. The fluorescence of 5,10-CH+-H4folate is unaffected by substrate. Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence. Quenching of FADH2 fluorescence is fully reversible upon dimer repair. The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis. Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗

PubMed1989

Mechanistic studies on phenylalanine hydroxylase from Chromobacterium violaceum. Evidence for the formation of an enzyme-oxygen complex.

Steady-state kinetic analysis of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum indicated that the enzyme follows a partially ordered reaction mechanism. The data suggested that oxygen is the first substrate to bind to the enzyme. This result was further supported by rapid-quench experiments in which the enzyme-oxygen complex was trapped to yield product. Additional support for the presence of an enzyme-oxygen complex was derived from magnetic susceptibility measurements of molecular oxygen in the presence and absence of cuprous phenylalanine hydroxylase. The magnetic susceptibility of dissolved oxygen decreased in the presence of the enzyme, supporting a direct oxygen-metal interaction.

Chromobacterium↗

PubMed1981

On the mechanism of action of phenylalanine hydroxylase.

The oxidation of 6-methyltetrahydropterin and tetrahydrobiopterin coupled to the formation of tyrosine by phenylalanine hydroxylase generates a precursor species to the quinonoid product that is tentatively identified as a 4a-hydroxy adduct based on its spectral similarity to the 4a-hydroxy-6-methyl-5-deazatetrahydropterin. The rate of appearance of this intermediate and that of tyrosine are equal and hydroxylase catalyzed in accord with the completion of the hydroxylation event. This observation, which confirms and extends an earlier one by Kaufman [Kaufman, S. (1975) in Chemistry and Biology of Pteridines (Pfleiderer, W., Ed.) p 291, Walter de Gruyter, Berlin], serves to link the reaction courses followed by pterin and pyrimidine cofactor analogues and supports the hypothesis that the 4a position is a site of O2 attachment. Thus, as expected, no prereduction of the enzyme was observed in anaerobic experiments utilizing stoichiometric amounts of enzyme and tetrahydropterin in the presence or absence of 1 mM phenylalanine. Activation of the hydroxylase by 1 mM lysolecithin leads to oxidation of the tetrahydropterin in the absence of phenylalanine. A ring-opened pyrimidine analogue of the tetrahydropterin, 2,5-diamino-4-[(meso-1-methyl-2-aminopropyl)amino]-6-hydroxypyrimidine, was studied to examine the possibility of tetrahydropterin ring opening in the enzymatic reaction prior to 4a-hydroxy adduct formation. However, no hydroxylase-catalyzed ring closure was observed.

Anaerobiosis↗

PubMed2002

Conformational changes in nitric oxide synthases induced by chlorzoxazone and nitroindazoles: crystallographic and computational analyses of inhibitor potency.

Nitric oxide is a key signaling molecule in many biological processes, making regulation of nitric oxide levels highly desirable for human medicine and for advancing our understanding of basic physiology. Designing inhibitors to specifically target one of the three nitric oxide synthase (NOS) isozymes that form nitric oxide from the L-Arg substrate poses a significant challenge due to the overwhelmingly conserved active sites. We report here 10 new X-ray crystallographic structures of inducible and endothelial NOS oxygenase domains cocrystallized with chlorzoxazone and four nitroindazoles: 5-nitroindazole, 6-nitroindazole, 7-nitroindazole, and 3-bromo-7-nitroindazole. Each of these bicyclic aromatic inhibitors has only one hydrogen bond donor and therefore cannot form the bidentate hydrogen bonds that the L-Arg substrate makes with Glu371. Instead, all of these inhibitors induce a conformational change in Glu371, creating an active site with altered molecular recognition properties. The cost of this conformational change is approximately 1-2 kcal, based on our measured constants for inhibitor binding to the wild-type and E371A mutant proteins. These inhibitors derive affinity by pi-stacking above the heme and replacing both intramolecular (Glu371-Met368) and intermolecular (substrate-Trp366) hydrogen bonds to the beta-sheet architecture underlying the active site. When bound to NOS, high-affinity inhibitors in this class are planar, whereas weaker inhibitors are nonplanar. Isozyme differences were observed in the pterin cofactor site, the heme propionate, and inhibitor positions. Computational docking predictions match the crystallographic results, including the Glu371 conformational change and inhibitor-binding orientations, and support a combined crystallographic and computational approach to isozyme-specific NOS inhibitor analysis and design.

Amino Acid Sequence↗

PubMed2004

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase.

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.

Binding Sites↗

PubMed2004

Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.

The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli.

Azides↗

PubMed2004

Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2.

An inherited form of diabetes, maturity-onset diabetes of the young type 3 (MODY3), results from mutations in the transcriptional activator, hepatocyte nuclear factor-1alpha (HNF1alpha). Transcription by HNF1alpha is stimulated by the bifunctional coactivator DCoH (dimerization cofactor of HNF1). Strikingly, an HNF1alpha deletion in mice causes more severe phenotypes than a DCoH deletion. It has been hypothesized that a DCoH homolog, DCoH2, partially complements the DCoH deletion. To test this idea, we determined the biochemical properties and the 1.6-A-resolution crystal structure of DCoH2. Like DCoH, DCoH2 forms a tetramer, displays pterin-4alpha-carbinolamine dehydratase activity, and binds HNF1alpha in vivo and in vitro. DCoH and DCoH2 adopt identical folds with structural differences confined largely to the protein surfaces and the tetramer interface. In contrast to the hyperstable DCoH tetramer, DCoH2 readily disproportionates and forms a 2:2 complex with HNF1 in vitro. Phylogenetic analysis reveals six major subfamilies of DCoH proteins, including unique DCoH and DCoH2 branches in metazoans. These results suggest distinct roles for DCoH and DCoH2. Differences in conserved surface residues could mediate binding to different effectors. We propose that HNF1alpha binding kinetics may distinguish regulation by DCoH2, under thermodynamic control, from regulation by DCoH, under kinetic control.

Amino Acid Sequence↗

PubMed2006

Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation.

In human methylenetetrahydrofolate reductase (MTHFR) the Ala222Val (677C-->T) polymorphism encodes a heat-labile gene product that is associated with elevated levels of homocysteine and possibly with risk for cardiovascular disease. Generation of the equivalent Ala to Val mutation in Escherichia coli MTHFR, which is 30% identical to the catalytic domain of the human enzyme, creates a protein with enhanced thermolability. In both human and E. coli MTHFR, the A --> V mutation increases the rate of dissociation of FAD, and in both enzymes, loss of FAD is linked to changes in quaternary structure [Yamada, K., Chen, Z., Rozen, R., and Matthews, R. G. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14853-14858; Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G., and Ludwig, M. L. (1999) Nat. Struct. Biol. 6, 359-365]. Folates have been shown to protect both human and bacterial enzymes from loss of FAD. Despite its effect on affinity for FAD, the A --> V mutation is located at the bottom of the (betaalpha)(8) barrel of the catalytic domain in a position that does not contact the bound FAD prosthetic group. Here we report the structures of the Ala177Val mutant of E. coli MTHFR and of its complex with the 5,10-dideazafolate analogue, LY309887, and suggest mechanisms by which the mutation may perturb FAD binding. Helix alpha5, which immediately precedes the loop bearing the mutation, carries several residues that interact with FAD, including Asn168, Arg171, and Lys172. In the structures of the mutant enzyme this helix is displaced, perturbing protein-FAD interactions. In the complex with LY309887, the pterin-like ring of the analogue stacks against the si face of the flavin and is secured by hydrogen bonds to residues Gln183 and Asp120 that adjoin this face. The direct interactions of bound folate with the cofactor provide one mechanism for linkage between binding of FAD and folate binding that could account in part for the protective action of folates. Conformation changes induced by folate binding may also suppress dissociation of FAD.

Alanine↗

PubMed1996

Putative human blue-light photoreceptors hCRY1 and hCRY2 are flavoproteins.

Recently, a human cDNA clone with high sequence homology to the photolyase/blue-light photoreceptor family was identified. The putative protein encoded by this gene exhibited a strikingly high (48% identity) degree of homology to the Drosophila melanogaster (6-4) photolyase [Todo et al. (1996) Science 272, 109-112]. We have now identified a second human gene whose amino acid sequence displays 73% identity to the first one and have named the two genes CRY1 and CRY2, respectively. The corresponding proteins hCRY1 and hCRY2 were purified and characterized as maltose-binding fusion proteins. Similar to other members of the photolyase/blue-light photoreceptor family, both proteins were found to contain FAD and a pterin cofactor. Like the plant blue-light photoreceptors, both hCRY1 and hCRY2 lacked photolyase activity on the cyclobutane pyrimidine dimer and the (6-4) photoproduct. We conclude that these newly discovered members of the photolyase/photoreceptor family are not photolyases and instead may function as blue-light photoreceptors in humans.

Amino Acid Sequence↗