Ligand trans influence governs conformation in cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme. Reductive methylation with flavodoxin and adenosylmethionine returns the enzyme to an active methylcobalamin state. At different points during the reaction cycle, the coordination state of the cobalt of the cobalamin changes. The imidazole side chain of His759 coordinates to cobalamin in a "His-on" state and dissociates to produce a "His-off" state. The His-off state has been associated with a conformation of MetH that is poised for reactivation of cobalamin by reductive methylation rather than catalysis. Our studies on cob(III)alamins bound to MetH, specifically aqua-, methyl-, and n-propylcobalamin, show a correlation between the accessibility of the reactivation conformation and the order of the established ligand trans influence. The trans influence also controls the affinity of MetH in the cob(III)alamin form for flavodoxin. Flavodoxin, which acts to shift the conformational equilibrium toward the reactivation conformation, binds less tightly to MetH when the cob(III)alamin has a strong trans ligand and therefore has less positive charge on cobalt. These results are compared to those for cob(II)alamin MetH, illustrating that access to the reactivation conformation is governed by the net charge on the cobalt as well as the trans influence in cob(III)alamins.     

Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269

The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S-adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure-based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80 degrees C, where the specific activity of the purified protein is approximately 15% of that of E. coli methionine synthase (MetH) at 37 degrees C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.     

Protein interactions in the human methionine synthase-methionine synthase reductase complex and implications for the mechanism of enzyme reactivation

Methionine synthase (MS) is a cobalamin-dependent enzyme. It transfers a methyl group from methyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate. On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH), human MS comprises four discrete functional modules that bind from the N- to C-terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (AdoMet). The C-terminal activation domain also interacts with methionine synthase reductase (MSR), a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin. We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR. We show that the MS activation domain interacts directly with the FMN-binding domain of MSR. Binding is weakened at high ionic strength, emphasizing the importance of electrostatic interactions at the protein-protein interface. Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction. Chemical cross-linking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain). The activation domain and isolated FMN-domain form a 1:1 complex, but a 1:2 complex is formed with activation domain and MSR. The midpoint reduction potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex, implying that electron transfer to cob(II)alamin is endergonic. The kinetics of electron transfer in MSR and the MSR-activation domain complex are similar. Our studies indicate (i) conserved binding determinants, but differences in protein stoichiometry, between human MS and bacterial MetH in complex formation with redox partners; (ii) a substantial endergonic barrier to electron transfer in the reactivation complex; and (iii) a lack of control on the thermodynamics and kinetics of electron transfer in MSR exerted by complex formation with activation domain. The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR-MS complex.     

Probing the role of the histidine 759 ligand in cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH.     

Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.     

Participation of cob(I) alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis

The kinetic mechanism of the reaction catalyzed by cobalamin-dependent methionine synthase from Escherichia coli K12 has been investigated by both steady-state and pre-steady-state kinetic analyses. The reaction catalyzed by methionine synthase involves the transfer of a methyl group from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The postulated reaction mechanism invokes an initial transfer of the methyl group to the enzyme to generate enzyme-bound methylcobalamin and tetrahydrofolate. Enzyme-bound methylcobalamin then donates its methyl group to homocysteine to generate methionine and cob(I)alamin. The key questions that were addressed in this study were the following: (1) Does the reaction involve a sequential or ping-pong mechanism? (2) Is enzyme-bound cob(I)alamin a kinetically competent intermediate? (3) If the reaction does involve a sequential mechanism, what is the nature of the "free" enzyme to which the substrates bind; i.e., is the prosthetic group in the cob(I)alamin or methylcobalamin state? Both the steady-state and rapid reaction studies were conducted at 25 degrees C under anaerobic conditions. Initial velocity analysis under steady-state conditions revealed a family of parallel lines suggesting either a ping-pong mechanism or an ordered sequential mechanism. Steady-state product inhibition studies provided evidence for an ordered sequential mechanism in which the first substrate to bind is methyltetrahydrofolate and the last product to be released is tetrahydrofolate. Pre-steady-state kinetic studies were then conducted to determine the rate constants for the various reactions. Enzyme-bound cob(I)alamin was shown to react very rapidly with methyltetrahydrofolate (with an observed rate constant of 250 s-1 versus a turnover number under maximal velocity conditions of 19 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R.T., & Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766. Fujii, K., & Huennekens, F.M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer in human methionine synthase reductase studied by stopped-flow spectrophotometry

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s(-1)) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s(-1)) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s(-1)) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-2H]NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP+. The observed rate constant of reverse hydride transfer increases hyperbolically with NADP+ concentration with the FAD domain. The stopped-flow kinetic data, in conjunction with the reported redox potentials of the flavin cofactors for MSR [Wolthers, K. R., Basran, J., Munro, A. W., and Scrutton, N. S. (2003) Biochemistry, 42, 3911-3920], are used to define the mechanism of electron transfer for the reductive half-reaction of MSR. Comparisons are made with similar stopped-flow kinetic studies of the structurally related enzymes cytochrome P450 reductase and nitric oxide synthase.     

Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study

The mechanism of reductive methylation of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13) has been investigated by electron paramagnetic resonance (EPR) spectroelectrochemistry. The enzyme as isolated is inactive, and its UV/visible absorbance and EPR spectra are characteristic of cob(II)alamin. There is an absolute requirement for catalytic amounts of AdoMet and a reducing system for the formation and maintenance of active enzyme during in vitro turnover. The midpoint potentials of the enzyme-bound cob(II)alamin/cob(I)alamin and cob(III)alamin/cob(II)alamin couples have been determined to be -526 +/- 5 and +273 +/- 4 mV (versus the standard hydrogen electrode), respectively. The presence of either CH3-H4folate or AdoMet shifts the equilibrium distribution of cobalamin species observed during reduction by converting cob(I)alamin to methylcobalamin. The magnitude of these shifts is however vastly different, with AdoMet lowering the concentration of cob(II)alamin at equilibrium by a factor of at least 3 X 10(7), while CH3-H4folate lowers it by a factor of 19. These studies of coupled reduction/methylation reactions elucidate the absolute requirement for AdoMet in the in vitro assay system, in which the ambient potential is approximately -350 mV versus the standard hydrogen electrode. At this potential, the equilibrium distribution of cobalamin in the presence of CH3-H4folate would be greatly in favor of the cob(II)alamin species, whereas in the presence of AdoMet the equilibrium favors methylated enzyme. In these studies, a base-on form of cob(II)alamin in which the dimethylbenzimidazole substituent of the corrin ring is the lower axial ligand for the cobalt has been observed for the first time on methionine synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro inactivation of methionine synthase by nitrous oxide

Nitrous oxide (N2O) is commonly used as an anesthetic agent. Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates. We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O. Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system. Reisolation of the inactivated E. coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation. However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin. We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical. Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity.     

Halomethane:bisulfide/halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source.

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH(3)Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH(3)Cl; then it catalyzed methyl transfer from CH(3)Cl, CH(3)Br, or CH(3)I to the following acceptor ions (in order of decreasing efficacy): I(-), HS(-), Cl(-), Br(-), NO(2)(-), CN(-), and SCN(-). Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N(2)O, and Hg(2+) to affect the methyltransferase suggests significant differences. During CH(3)Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS(-), a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH(3)Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899-2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide

Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH(3)-H(4)folate) and homocysteine, with the enzyme-bound cobalamin serving as an intermediary in the methyl transfers. An MetH fragment comprising residues 2-649 contains modules that bind and activate CH(3)-H(4)folate and homocysteine and catalyze methyl transfers to and from exogenous cobalamin. Comparison of the rates of reaction of cobalamin, which contains a dimethylbenzimidazole nucleotide coordinated to the cobalt in the lower axial position, and cobinamide, which lacks the dimethylbenzimidazole nucleotide, allows assessment of the degree of stabilization the dimethylbenzimidazole base provides for methyl transfer between CH(3)-H(4)folate bound to MetH(2-649) and exogenous cob(I)alamin. When the reactions of cob(I)alamin or cob(I)inamide with CH(3)-H(4)folate are compared, the observed second-order rate constants are 2.7-fold faster for cob(I)alamin; in the reverse direction, methylcobinamide reacts 35-fold faster than methylcobalamin with enzyme-bound tetrahydrofolate. These measurements can be used to estimate the influence of the dimethylbenzimidazole ligand on both the thermodynamics and kinetics of methyl transfer between methyltetrahydrofolate and cob(I)alamin or cob(I)inamide. The free energy change for methyl transfer from CH(3)-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes approximately 0.6 kcal/mol of stabilization for the forward reaction and approximately 2.2 kcal/mol of destabilization for the reverse reaction. Binding of methylcobalamin to full-length methionine synthase is accompanied by ligand substitution, and switching between "base-on" and "base-off" states of the cofactor has been demonstrated [Bandarian, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8156-8163]. The present results disfavor a major role for such switching in catalysis of methyl transfer, and are consistent with the hypothesis that the primary role of the ligand triad in methionine synthase is controlling the distribution of enzyme conformations during catalysis.     

Electron transfer in the substrate-dependent suicide inactivation of lysine 5,6-aminomutase

The lysine 5,6-aminomutase (5,6-LAM) purified from Clostridium sticklandii was found to undergo rapid inactivation in the absence of the activating enzyme E(2) and ATP. In the presence of substrate, inactivation was also seen for the recombinant 5,6-LAM. This adenosylcobalamin-dependent enzyme is postulated to generate cob(II)alamin and the 5'-deoxyadenosyl radical through enzyme-induced homolytic scission of the Co-C bond. However, the products cob(III)alamin and 5'-deoxyadenosine were observed upon inactivation of 5,6-LAM. Cob(III)alamin production, as monitored by the increase in A(358), proceeds at the same rate as the loss of enzyme activity, suggesting that the activity loss is related to the adventitious generation of cob(III)alamin during enzymatic turnover. The cleavage of adenosylcobalamin to cob(III)alamin is accompanied by the formation of 5'-deoxyadenosine at the same rate, and the generation of cob(III)alamin proceeds at the same rate both aerobically and anaerobically. Suicide inactivation requires the presence of substrate, adenosylcobalamin, and PLP. We have ruled out the involvement of either the putative 5'-deoxyadenosyl radical or dioxygen in suicide inactivation. We have shown that one or more reaction intermediates derived from the substrate or/and the product, presumably a radical, participate in suicide inactivation of 5,6-LAM through electron transfer from cob(II)alamin. Moreover, L-lysine is found to be a slowly reacting substrate, and it induces inactivation at a rate similar to that of D-lysine. The alternative substrate beta-lysine induces inactivation at least 25 times faster than DL-lysine. The inactivation mechanism is compatible with the radical isomerization mechanism proposed to explain the action of 5,6-LAM.     

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)相似文献   

Isolation, cloning, mapping, and nucleotide sequencing of the gene encoding flavodoxin in Escherichia coli.

The flavodoxins constitute a highly conserved family of small, acidic electron transfer proteins with flavin mononucleotide prosthetic groups. They are found in prokaryotes and in red and green algae, where they provide electrons at low potentials for the reduction of nitrogen by nitrogenase, for the light-dependent reduction of NADP+ in photosynthesis, and for the reduction of sulfite. Proteins with the physical characteristics of flavodoxins have been implicated in the reductive activation of pyruvate formate-lyase and cobalamin-dependent methionine synthase in Escherichia coli. We have purified flavodoxin to homogeneity from E. coli, determined its N-terminal amino acid sequence, and used this sequence to construct a 64-fold degenerate oligonucleotide probe for the flavodoxin gene. Because the phenotype of a flavodoxin mutant is not known, we used this degenerate probe to screen the phages of the Kohara library and identified two phages, with inserts mapping at approximately 16 min, that hybridized to the probe. The flavodoxin gene, designated fldA, was subcloned from the DNA in the overlap region of these two clones. The deduced amino acid sequence, determined by nucleotide sequencing of the flavodoxin gene, shows strong homology with flavodoxins from nitrogen-fixing bacteria and cyanobacteria. The fldA gene maps at 15.9 min on the E. coli chromosome and is transcribed in a counterclockwise direction.     

Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH₃Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH₃Cl; then it catalyzed methyl transfer from CH₃Cl, CH₃Br, or CH₃I to the following acceptor ions (in order of decreasing efficacy): I⁻, HS⁻, Cl⁻, Br⁻, NO₂⁻, CN⁻, and SCN⁻. Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N₂O, and Hg²⁺ to affect the methyltransferase suggests significant differences. During CH₃Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS⁻, a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH₃Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899–2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.     

Mechanism of conversion of human apo- to holomethionine synthase by various forms of cobalamin.

Methionine synthase catalyzes the conversion of N5-methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine. Methylcobalamin (Me-Cbl) is tightly bound to methionine synthase and is required for enzymatic activity. When added to crude tissue homogenates, Me-Cbl stimulates methionine synthase but similar stimulation is observed with hydroxocobalamin, cyanocobalamin (CN-Cbl), and adenosyl-Cbl, although the mechanisms involved are unknown. We prepared human apomethionine synthase and studied its activation in the presence of [14C]CN-Cbl and [14CH3]Me-Cbl with concentrations of 2-mercaptoethanol ranging from 0.15 to 100 mM. We observed that the removal of the labeled upper axial ligands from CN-Cbl and Me-Cbl both paralleled the activation of human apomethionine synthase. Spectral studies employing CN-Cbl and Me-Cbl showed that both forms of Cbl must be converted to Cob(II)alamin before they can bind to human apomethionine synthase and convert it to its activated holoenzyme form. Studies with 14 different Cbl analogues with alterations in various portions of the corrin ring and the nucleotide showed that all of the analogues were able to fully activate human methionine synthase when they were reduced with 2-mercaptoethanol. Full activation occurred at lower concentrations of many of the Cbl analogues than occurred with Cbl itself. We conclude that Me-Cbl and other forms of Cob(III)alamin do not bind to human apomethionine synthase and that all must first be reduced to Cob(II)alamin before such binding can occur. The fact that human methionine synthase shows little absolute specificity for alterations in various portions of the Cbl molecule suggests that the potent inhibition of mammalian methionine synthase activity observed in vivo with various Cbl analogues is due to inhibition of intracellular Cbl transport or to inhibition of the enzymatic formation of Cob(II)alamin rather than to direct inhibition of mammalian methionine synthase itself.     

Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri -- substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin.

Methyl-coenzyme M formation from coenzyme M and methanol in Methanosarcina barkeri is catalysed by an enzyme system composed of three polypeptides MtaA, MtaB and MtaC, the latter of which harbours a corrinoid prosthetic group. We report here that MtaC can be substituted by free cob(I)alamin which is methylated with methanol in an MtaB-catalysed reaction and demethylated with coenzyme M in an MtaA-catalysed reaction. Methyl transfer from methanol to coenzyme M was found to proceed at a relatively high specific activity at micromolar concentrations of cob(I)alamin. This finding was surprising because the methylation of cob(I)alamin catalysed by MtaB alone and the demethylation of methylcob(III)alamin catalysed by MtaA alone exhibit apparent Km for cob(I)alamin and methylcob(III)alamin of above 1 mm. A possible explanation is that MtaA positively affects the MtaB catalytic efficiency and vice versa by decreasing the apparent Km for their corrinoid substrates. Activation of MtaA by MtaB was methanol-dependent. In the assay for methanol:coenzyme M methyltransferase activity cob(I)alamin could be substituted by cob(I)inamide which is devoid of the nucleotide loop. Substitution was, however, only possible when the assays were supplemented with imidazole: approximately 1 mm imidazole being required for half-maximal activity. Methylation of cob(I)inamide with methanol was found to be dependent on imidazole but not on the demethylation of methylcob(III)inamide with coenzyme M. The demethylation reaction was even inhibited by imidazole. The structure and catalytic mechanism of the MtaABC complex are compared with the cobalamin-dependent methionine synthase.