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.     

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.     

Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase

Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor. The cobalamin cofactor serves as an intermediate carrier of the methyl group from methyltetrahydrofolate to homocysteine. In the two half-reactions that comprise turnover for MetH, the cobalamin is alternatively methylated by methyltetrahydrofolate and demethylated by homocysteine to form methionine. Upon binding to the protein, the usual dimethylbenzimidazole ligand is replaced by the imidazole side chain of His759 [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674]. Despite the ligand replacement that accompanies binding of cobalamin to the holo-MetH protein, a MetH(2-649) fragment of methionine synthase that contains the regions that bind homocysteine and methyltetrahydrofolate utilizes exogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle. However, the interactions of MetH(2-649) with endogenous cobalamin are first order in cobalamin, while the half-reactions catalyzed by the holoenzyme are zero order in cobalamin, so rate constants for reactions of bound and exogenous cobalamins cannot be compared. In this paper, we investigate the catalytic rate enhancements generated by binding cobalamin to MetH after dividing the protein in half and reacting MetH(2-649) with a second fragment, MetH(649-1227), that harbors the cobalamin cofactor. The second-order rate constant for demethylation of methylcobalamin by Hcy is elevated 60-fold and that for methylation of cob(I)alamin is elevated 120-fold. Thus, binding of cobalamin to MetH is essential for efficient catalysis.     

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.     

Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase

N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases and S-adenosyl-l-methionine (AdoMet)-dependent N-methyltransferases. The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes. Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site. The role of four conserved motifs (I, (2085)VLEIGTGSGMIL; II/Y, (2105)SYVGLDPS; IV, (2152)DLVVFNSVVQYFTPPEYL; and V, (2194)ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied. Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding. Further shortening close to motif I resulted in loss of binding activity. Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity. Point mutations converting the conserved Tyr(223) (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to approximately 70%, indicating that Tyr(223) is important for AdoMet binding and that the aromatic Tyr(223) may be crucial for AdoMet binding in N-methylpeptide synthetases.     

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.     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

Molecular dissection of the S-adenosylmethionine-binding site of phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a quatrotopic membrane protein that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three sequential methylation reactions. Analysis of mice lacking a functional PEMT gene revealed a severe reduction in plasma homocysteine levels. Homocysteine is generated by the hydrolysis of S-adenosylhomocysteine, which is also a product of the PEMT reaction. To gain insight into the PEMT transmethylation reaction and the mechanism by which PEMT regulates homocysteine levels, we sought to define residues that are required for binding of the methyl group donor, S-adenosylmethionine (AdoMet). Bioinformatic analysis of the predicted amino acid sequence of human PEMT identified two putative AdoMet-binding motifs (98GXG100 and 180EE181). Site-directed mutagenesis experiments demonstrated the requirement for the conserved motifs in PEMT specific activity. Analysis of the AdoMet binding ability of mutant recombinant PEMT derivatives established that residues Gly100 and Glu180 are essential for binding of the AdoMet moiety. A significantly elevated KD with respect to AdoMet is observed following conservative mutagenesis of residues Gly98 (400 pmol) and Glu181 (666.7 pmol), relative to the unmodified enzyme (303.1 pmol), suggesting that these residues also participate in AdoMet binding. A model positions two separate AdoMet-binding motifs of PEMT in close proximity at the external leaflet of the endoplasmic reticulum membrane.     

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.     

Structure determination of fibrillarin from the hyperthermophilic archaeon Pyrococcus furiosus

The methyltransferase fibrillarin is the catalytic component of ribonucleoprotein complexes that direct site-specific methylation of precursor ribosomal RNA and are critical for ribosome biogenesis in eukaryotes and archaea. Here we report the crystal structure of a fibrillarin ortholog from the hyperthermophilic archaeon Pyrococcus furiosus at 1.97A resolution. Comparisons of the X-ray structures of fibrillarin orthologs from Methanococcus jannashii and Archaeoglobus fulgidus reveal nearly identical backbone configurations for the catalytic C-terminal domain with the exception of a unique loop conformation at the S-adenosyl-l-methionine (AdoMet) binding pocket in P. furiosus. In contrast, the N-terminal domains are divergent which may explain why some forms of fibrillarin apparently homodimerize (M. jannashii) while others are monomeric (P. furiosus and A. fulgidus). Three positively charged amino acids surround the AdoMet-binding site and sequence analysis indicates that this is a conserved feature of both eukaryotic and archaeal fibrillarins. We discuss the possibility that these basic residues of fibrillarin are important for RNA-guided rRNA methylation.     

Crystal structure of RlmM, the 2��O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2′O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM–AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2′O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.     

Structure and function of the antibiotic resistance-mediating methyltransferase AviRb from Streptomyces viridochromogenes

The emergence of antibiotic-resistant bacterial strains is a widespread problem in medical practice and drug design, and each case requires the elucidation of the underlying mechanism. AviRb from Streptomyces viridochromogenes methylates the 2'-O atom of U2479 of the 23S ribosomal RNA in Gram-positive bacteria and thus mediates resistance to the oligosaccharide (orthosomycin) antibiotic avilamycin. The structure of AviRb with and without bound cofactor S-adenosyl-L-methionine (AdoMet) was determined, showing that it is a homodimer belonging to the SpoU family within the SPOUT class of methyltransferases. The relationships within this class were analyzed in detail and, in addition, a novel fourth SpoU sequence fingerprint is proposed. Each subunit of AviRb consists of two domains. The N-terminal domain, being related to the ribosomal proteins L30 and L7Ae, is likely to bind RNA. The C-terminal domain is related to all SPOUT methyltransferases, and is responsible for AdoMet-binding, catalysis and dimerization. The cofactor binds at the characteristic knot of the polypeptide in an unusually bent conformation. The transferred methyl group points to a broad cleft formed with the L30-type domain of the other subunit. Measurements of mutant activity revealed four important residues responsible for catalysis and allowed the modeling of a complex between AviRb and the RNA target. The model includes a specificity pocket for uracil but does not contain a base for deprotonating the 2'-O atom of U2479 on methylation.     

Insights into catalysis by a knotted TrmD tRNA methyltransferase

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.     

Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition

tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to G(37) within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G(36)G(37) in the active site of TrmD and the catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD(2)-tRNA(2) complex, which provides insights into recognition of the general tRNA structure by TrmD.     

Crystal structures of the Apo and Holo form of rat catechol-O-methyltransferase

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is a monomeric enzyme that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the phenolic oxygen of substituted catechols. Although the inhibitor recognition pattern and AdoMet site have already been studied crystallographically, structural information on the catalytic cycle of COMT has not yet been obtained. In this study, comparison of the co-factor and inhibitor-bound structures revealed that the Apo form of COMT shows a conformational change and there was no cleft corresponding to the AdoMet-binding site; the overall structure was partially open form and the substrate recognition site was not clearly defined. The Holo form of COMT was similar to the quaternary structure except for the β6–β7 and α2–α3 ligand recognition loops. These conformational changes provide a deeper insight into the structural events occurring in reactions catalyzed by AdoMet.     

Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region

Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.     

Crystal structure and solution characterization of the activation domain of human methionine synthase

Human methionine synthase (hMS) is a multidomain cobalamin-dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 A crystal structure of the C-terminal activation domain of hMS. The structure is C-shaped with the core comprising mixed alpha and beta regions, dominated by a twisted antiparallel beta sheet with a beta-meander region. These features, including the positions of the active-site residues, are similar to the activation domain of Escherichia coli cobalamin-dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S-adenosyl-methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild-type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 3(10)-containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex.     

Inhibition of catechol-O-methyltransferase by N-(3,4-dihydroxyphenyl) maleimide

Catechol-O-methyltransferase (COMT) is inhibited rapidly and irreversibly by N-(3,4-dihydroxyphenyl) maleimide. S-adenosylmethionine (AdoMet) and magnesium ions protect the enzyme from inactivation by this compound, but no protection is observed by the catechol substrate. However, the corresponding succinimide analogue shows a reversible inhibition of COMT, which is competitive with pyrocatecholphthalein and non-competitive with AdoMet. Amino-group reagents also inhibit COMT and this inhibition is protected by AdoMet, suggesting that sulphydryl and amino groups essential for activity are located in an AdoMet-binding site on COMT. The maleimide derivative may be considered to be an active-site directed inhibitor.     

Protonation state of methyltetrahydrofolate in a binary complex with cobalamin-dependent methionine synthase

N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl group attached to a tertiary amine is a reaction without an obvious precedent in bioorganic chemistry. Activation of CH(3)-H(4)folate by protonation prior to transfer of the methyl group has been the favored mechanism. Protonation at N5 would lead to formation of an aminium cation, and quaternary amines such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl groups to cob(I)alamin. Because CH(3)-H(4)folate is an enamine, protonation could occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used (13)C distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy to infer that CH(3)-H(4)folate in aqueous solution protonates at N5, not on carbon. CH(3)-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this protonation could occur either upon formation of the binary enzyme-CH(3)-H(4)folate complex or later in the reaction mechanism. Protonation at N5 is accompanied by substantial changes in the visible absorbance spectrum of CH(3)-H(4)folate. We have measured the spectral changes associated with binding of CH(3)-H(4)folate to a catalytically competent fragment of MetH over the pH range from 5.5 to 8.5. These studies indicate that CH(3)-H(4)folate is bound in the unprotonated form throughout this pH range and that protonated CH(3)-H(4)folate does not bind to the enzyme. Our observations are rationalized by sequence homologies between the folate-binding region of MetH and dihydropteroate synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha(8)beta(8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl transfer and suggest that the presence of the cobalamin cofactor is important for CH(3)-H(4)folate activation. We propose that protonation of N5 occurs after carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidative addition of Co(1+) to the N5-methyl bond to rationalize our results.     

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet [10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.