Crystal structures of CbiL, a methyltransferase involved in anaerobic vitamin B biosynthesis, and CbiL in complex with S-adenosylhomocysteine--implications for the reaction mechanism

During anaerobic cobalamin (vitamin B12) biosynthesis, CbiL catalyzes methylation at the C-20 position of a cyclic tetrapyrrole ring using S-adenosylmethionine as a methyl group source. This methylation is a key modification for the ring contraction process, by which a porphyrin-type tetrapyrrole ring is converted to a corrin ring through elimination of the modified C-20 and direct bonding of C-1 to C-19. We have determined the crystal structures of Chlorobium tepidum CbiL and CbiL in complex with S-adenosylhomocysteine (the S-demethyl form of S-adenosylmethionine). CbiL forms a dimer in the crystal, and each subunit consists of N-terminal and C-terminal domains. S-Adenosylhomocysteine binds to a cleft between the two domains, where it is specifically recognized by extensive hydrogen bonding and van der Waals interactions. The orientation of the cobalt-factor II substrate was modeled by simulation, and the predicted model suggests that the hydroxy group of Tyr226 is located in close proximity to the C-20 atom as well as the C-1 and C-19 atoms of the tetrapyrrole ring. These configurations allow us to propose a catalytic mechanism: the conserved Tyr226 residue in CbiL catalyzes the direct transfer of a methyl group from S-adenosylmethionine to the substrate through an S(N)2-like mechanism. Furthermore, the structural model of CbiL binding to its substrate suggests the axial residue coordinated to the central cobalt of cobalt-factor II.     

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 DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.     

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.     

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.     

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)     

Changing the DNA Recognition Specificity of the EcoDam DNA-(Adenine-N6)-Methyltransferase by Directed Evolution

EcoDam is an adenine-N6 DNA methyltransferase that methylates the GATC sites in the Escherichia coli genome. We have changed the target specificity of EcoDam from GATC to GATT by directed evolution, combining different random mutagenesis methods with restriction protection at GATT sites for selection and screening. By co-evolution of an enzyme library and a substrate library, we identified GATT as the best non-GATC site and discover a double mutation, R124S/P134S, as the first step to increase enzyme activity at GATT sites. After four generations of mutagenesis and selection, we obtained enzyme variants with new specificity for GATT. While the wild-type EcoDam shows no detectable activity at GATT sites in E. coli cells, some variants prefer methylation at GATT over GATC sites by about 10-fold in cells. In vitro DNA methylation kinetics carried out under single-turnover conditions using a hemimethylated GATC and a GATT oligonucleotide substrate confirmed that the evolved proteins prefer methylation of GATT sites to a similar degree. They show up to 1600-fold change in specificity in vitro and methylate the new GATT target site with 20% of the rate of GATC methylation by the wild-type enzyme, indicating good activity. We conclude that the new methyltransferases are fully functional in vivo and in vitro but show a new target-site specificity.     

Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine] methyltransferase (Dam) of bacteriophage T4.

Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA. Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites. We have determined that the damh mutation produces a single amino acid change (Pro126 to Ser126) in a region of homology (III) shared by three DNA-adenine methyltransferases; viz, T4 Dam, Escherichia coli Dam, and the DpnII modification enzyme of Streptococcus pneumoniae. We also describe another mutant, damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA. This mutation also alters a single amino acid (Phe127 to Val127). These results implicate homology region III as a domain involved in DNA sequence recognition. The effect of several different amino acids at residue 126 was examined by creating a polypeptide chain terminating codon at that position and comparing the methylation capability of partially purified enzymes produced in the presence of various suppressors. No enzyme activity is detected when phenylalanine, glutamic acid, or histidine is inserted at position 126. However, insertion of alanine, cysteine, or glycine at residue 126 produces enzymatic activity similar to Damh.     

Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue

The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-l-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methylation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. ENZYMES: Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. STRUCTURED DIGITAL ABSTRACT: ? Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) ? Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction).     

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

Phospholipid methyltransferase asymmetry in synaptosomal membranes

The sequential methylation of phosphatidylethanolamine to form phosphatidylcholine is carried out by two methyltransferases in rat brain synaptosomes. The first enzyme methylates phosphatidylethanolamine to form phosphatidylmonomethylethanolamine. The second enzyme methylates the monomethylated phospholipid two additional times, forming phosphatidylcholine. Experiments comparing the rate of methylation between intact and lysed synaptosomes indicate that synaptosomes accumulateS-adenosyl-l-methionine and that the first methylation takes place on the cytoplasmic side of the membrane. Studies comparing trypsin digestion of proteins in intact and lysed synaptosomes indicate that the first enzyme is localized on the cytoplasmic side of the membrane and the second enzyme faces the external surface. Phospholipase C hydrolyzed phosphatidylcholine formed by methylation, suggesting its localization in the external layer of the phospholipid bilayer. A mechanism for an enzyme-mediated flip-flop of phospholipids from the cytoplasmic side to the outer surface of the synaptosomal plasma membrane is presented.     

Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35 are insufficient on their own to support the methylation reaction. Here we use biochemical techniques in conjunction with heteronuclear/homonuclear nuclear magnetic resonance spectroscopy to define the RNA structures that are required for efficient methylation by RlmA(II). Progressive truncation of the rRNA substrate indicated that multiple contacts occur between RlmA(II) and nucleotides in stem-loops 33, 34 and 35. RlmA(II) appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar to that of the orthologous Gram-negative methyltransferase, RlmA(I) (formerly RrmA), which also interacts with hairpin 35, but methylates at the adjacent nucleotide G745.     

Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Partial purification and characterisation of S-adenosylmethionine:protein-histidine N-methyltransferase from rabbit skeletal muscle

A new class of protein methylase (S-adenosylmethionine:protein-histidine N-methyltransferase) which methylates histidine residues of protein substrates using S-adenosylmethionine as the methyl donor has been partially purified from rabbit skeletal muscle, 22-fold with a yield of 56%. The enzyme activity was monitored using denatured myofibrils from young rabbit skeletal muscle as the methyl acceptor protein substrate. The enzyme was localised in the myofibrillar fraction and myofibrils isolated in pure form represented the enzyme-substrate complex. The enzyme was solubilised in 0.275 M KCl. The methylase showed no requirement for any metal ion and has a pH optimum of 8.0. It was shown to require a reducing agent like mercaptoethanol for its activity. It was also shown that cardiac and skeletal muscle forms of actins obtained from different species serve as the efficient methyl acceptor protein substrates. Since the enzyme was found to methylate specifically the histidine residues of actin we propose to designate this new methylase as protein methylase IV, to distinguish it from the already known protein methylases I, II and III.     

Domain alternation switches B(12)-dependent methionine synthase to the activation conformation.

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that uses bound cobalamin as an intermediate methyl carrier. Major domain rearrangements have been postulated to explain how cobalamin reacts with three different substrates: homocysteine, methyltetrahydrofolate and S-adenosylmethionine (AdoMet). Here we describe the 3.0 A structure of a 65 kDa C-terminal fragment of MetH that spans the cobalamin- and AdoMet-binding domains, arranged in a conformation suitable for the methyl transfer from AdoMet to cobalamin that occurs during activation. In the conversion to the activation conformation, a helical domain that capped the cofactor moves 26 A and rotates by 63 degrees, allowing formation of a new interface between cobalamin and the AdoMet-binding (activation) domain. Interactions with the MetH activation domain drive the cobalamin away from its binding domain in a way that requires dissociation of the axial cobalt ligand and, thereby, provide a mechanism for control of the distribution of enzyme conformations.     

Synthesis of substrate analogs of methyltransferases in the vitamin B(12) biosynthetic pathway and characterization of their enzymatic products

The specificity toward substrate analogs of the first two methyltransferases in the vitamin B(12) biosynthetic pathway was probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and characterized, suggesting the same methylation sequence C-2>C-7>C-20 as for the natural substrate, uro'gen III. The results allow us to narrow down possible structural requirements concerning substrate recognition by the methyltransferase enzymes.     

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.