Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-L-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 A, beta = 104.97 degrees. Native data to a maximum resolution of 1.4 A were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

Isolation, cloning and expression of a multifunctional O-methyltransferase capable of forming 2,5-dimethyl-4-methoxy-3(2H)-furanone, one of the key aroma compounds in strawberry fruits.

Strawberry fruits contain an uncommon group of key aroma compounds with a 2,5-dimethyl-3(2H)-furanone structure. Here, we report on the methylation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) by a S-adenosyl-L-methionine dependent O-methyltransferase, the cloning of the corresponding cDNA and characterization of the encoded protein. Northern-hybridization indicated that the Strawberry-OMT specific transcripts accumulated during ripening in strawberry fruits and were absent in root, petiole, leaf and flower. The protein was functionally expressed in E. coli and exhibited a substrate specificity for catechol, caffeic acid, protocatechuic aldehyde, caffeoyl CoA and DMHF. A common structural feature of the accepted substrates was a o-diphenolic structure also present in DMHF in its dienolic tautomer. FaOMT is active as a homodimer and the native enzyme shows optimum activity at pH 8.5 and 37 degrees C. It does not require a cofactor for enzymatic activity. Due to the expression pattern of FaOMT and the enzymatic activity in the different stages of fruit ripening we suppose that FaOMT is involved in lignification of the achenes and the vascular bundles in the expanding fruit. In addition, it is concluded that the Strawberry-OMT plays an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF.

Purification and characterization of protein methylase II from Helicobacter pylori.

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.

Purification and characterization of a O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine from Vitis vinifera L. (cv. Cabernet Sauvignon).

An S-adenosyl-L-methionine-dependent O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatography on DEAE-Sepharose FF, Ether-5PW, hydroxyapatite, G2000SW(XL), and DEAE-5PW. The relative molecular mass of the native enzyme estimated on gel permeation chromatography was 85 kDa, and the subunit molecular mass was estimated to be 41 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme also methylates caffeic acid. The Vmax for IBHP and caffeic acid were 0.73 and 175 pkatals/mg, respectively, and the respective Km for IBHP and caffeic acid were 0.30 and 0.032 mm. The optimum pH for IBHP (8.5) was different from that for caffeic acid (7.5).

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

An endogenous proteinacious inhibitor for S-adenosyl-L-methionine-dependent transmethylation reactions; identification of S-adenosylhomocystein as an integral part.

A proteinacious inhibitor with a molecular weight of 1,600 Da which inhibits S-adenosyl-L-methionine-dependent transmethylation reactions was purified from porcine liver to homogeneity by procedures including boiling, Sephadex G-25 column chromatography and repeated HPLC. Employing both Nuclear Magnetic Resonance (NMR) and Fast Atom Bombardment-Mass (FAB-Mass) spectroscopy, S-adenosylhomocysteine was conclusively identified as an integral part of the inhibitor. The purified S-adenosylhomocysteine was competitive with S-adenosyl-L-methionine with Ki value of 6.3x10(-6) M towards protein methylase II.

Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain.

We have recently described a novel protein carboxyl methylation system that results in the reversible modification of a 36-kDa polypeptide component of a 178-kDa protein in the cytosol of a variety of eucaryotic cells. This reaction, catalyzed by a cytosolic 40-kDa methyl-transferase, results in the methyl esterification of the alpha-carboxyl group of the C-terminal leucine residue. We have now purified the major methylated 36-kDa polypeptide from bovine brain. N-terminal sequence analysis of a tryptic fragment of this polypeptide revealed identity to the catalytic subunit of protein phosphatase 2A. This enzyme exists in the cell predominantly as a trimeric 151-kDa native species containing the 36-kDa catalytic polypeptide that terminates in a leucine residue. We then fractionated bovine brain cytosolic extracts to separate the major phosphatase isoforms 2A1 and 2A2 and found that both could be methylated by a partially purified preparation of the methyltransferase. A synthetic C-terminal octapeptide based on the sequence of the 36-kDa catalytic subunit is neither a substrate nor an inhibitor of this methyltransferase, suggesting that this enzyme recognizes aspects of the tertiary and/or quaternary structure of the native phosphatase. Because this modification reaction is readily reversible in extracts, it may represent a novel strategy of the cell to modulate the function of this protein phosphatase.

Methyl esterification of C-terminal leucine residues in cytosolic 36-kDa polypeptides of bovine brain. A novel eucaryotic protein carboxyl methylation reaction.

Incubation of cytosolic extracts of bovine brain with S-adenosyl[methyl-3H]methionine results in the predominant [3H]methyl esterification of a 36-kDa polypeptide. This reaction appears to be distinct from any of the three known types of protein carboxyl methylation reactions previously established. We show here that the methylated 36-kDa polypeptide is a component of a cytosolic protein with a native molecular mass estimated at 178 kDa by gel filtration chromatography. The methyl group is not stable on the protein and is lost as [3H]methanol with a half-life of about 180 min at pH 7.0, 37 degrees C. The methyltransferase responsible for this reaction is a cytosolic protein with a native molecular mass of about 40 kDa that is readily separated from the well described protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77). The methyl ester linkage is cleaved by carboxypeptidase Y, suggesting that the 36-kDa polypeptide is methylated on its C-terminal carboxyl group. Extensive digestion of gel-purified 3H-methylated 36-kDa polypeptide with trypsin and leucine aminopeptidase results in a radioactive product that co-chromatographs with authentic L-leucine methyl ester in reverse phase high performance liquid chromatography (HPLC), thin layer chromatography, thin layer electrophoresis, and high resolution-sulfonated polystyrene cation-exchange chromatography. Additionally, the o-phthalaldehyde/beta-mercaptoethanol-derived isoindole derivative of the 3H digestion product co-migrates on HPLC with the corresponding isoindole for L-leucine methyl ester. We demonstrate that a similar methylation system is present in yeast Saccharomyces cerevisiae but not in the bacterium Escherichia coli. These results provide evidence for a new type of reversible posttranslational modification reaction that may function to modulate the activities of its methyl-accepting substrates.

Purification and characterization of a membrane-bound protein carboxyl methyltransferase from rat kidney cortex.

Class II protein carboxyl methyltransferases (EC 2.1.1.77) are known to exist predominantly in a soluble form in all cells studied so far. These enzymes have been purified to homogeneity from the cytosols of many mammalian tissues but not from membranes. We describe here the purification to apparent homogeneity of a membrane-associated protein carboxyl methyltransferase from the brush border membrane of rat kidney. The enzyme was purified by fast protein liquid chromatography on Superdex 75 and Mono-Q and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of a single 27,300 polypeptide. The purified enzyme recognizes exogenous substrate proteins such as ovalbumin and gamma-globulins as well as synthetic peptides containing a L-isoaspartyl residue but not a synthetic peptide containing a farnesylated C-terminal cysteine (S-farnesyl-LARYKC). The Km for S-adenosyl-L-methionine with ovalbumin as the substrate is 1.5 microM and the purified enzyme is sensitive to inhibition by S-adenosyl-L-homocysteine (Ki = 0.3 microM). Peptide map obtained after Staphylococcus aureus V8 protease digestion of brush border membrane protein carboxyl methyltransferase showed a fragmentation pattern that was identical to that obtained for a soluble protein carboxyl methyltransferase purified according to the same procedure, indicating a high degree of homology. These results support the notion that class II protein carboxyl methyltransferases are not restricted to a cytosolic localization and show that the membrane-bound form of this enzyme shares many characteristics with known cytosolic protein carboxyl methyltransferases.

Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase.

Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl-accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staphylococcal V8 proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu-82-Asn-83 and Glu-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.

Asparaginyl deamidation-methylation of rat ventricular myosin light chains.

Spontaneous asparaginyl deamidation can produce damage to cytoskeletal proteins, and may lead to their targeting for subsequent rapid intracellular breakdown or repair. To test if myofibrillar proteins are subject to spontaneous deamidation damage in vitro, purified rat ventricular myosin light chain 1 (MLC1v) and phosphorylatable myosin light chain 2 (MPLC2v) were incubated (37 degrees C, 4 h, pH 2-11), and tested as substrates for human erythrocyte and rat cardiac protein carboxyl methyltransferase (PCMT). PCMT catalyzes the transfer of a methyl group from [3H-methyl] S-adenosyl methionine to deamidated asparaginyl residues and altered aspartyl residues on damaged proteins. MLC1v and MPLC2v underwent extensive incubation damage at neutral and alkaline pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed 3H-incorporation into MLC1v, MPLC2v, and a Mr = 14,000 polypeptide. 3H-methylated, CNBr-cleavage fragments of PCMT-methylated light chains were then separated by reverse-phase high performance liquid chromatography, and sequenced by automated Edman degradation. The major 3H-labeled peptide of the Mr = 14,000 protein proved homologous to residues 84 to 104 of rat MPLC2v, with a proposed deamidation site at Asn99-Ala100. The major 3H-labeled peptide from MLC1v proved homologous to residues 73 to 111 of rat cardiac MLC1v, with a proposed deamidation site at Asn108-Ser109. These results indicate that both myofibrillar protein subunits undergo selective non-enzymatic degradation at neutral and alkaline pH, resulting in the formation of methyl acceptor sites for human erythrocyte and rat cardiac PCMT. PCMT-catalyzed methylation of ventricular myosin light chains may be important in the repair, or subsequent proteolysis of these long-lived structural proteins of the myofibril.

Primary structure of rat brain protein carboxyl methyltransferase deduced from cDNA sequence.

Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambda gt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells.

Two distinct protein carboxyl methyltransferases (PCM) were identified in the electric organ of Torpedo ocellata. They were separated from each other in the active form by means of nondenaturing gel electrophoresis and by p-(chloromercuri)benzoate-agarose chromatography, and were individually identified by specific polyclonal antibodies. The existence of at least two distinct PCMs in eucaryotic cells raises the possibility that these enzymes are involved in distinct transmethylation reactions.

Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes.

We have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. We found sequence identify or probable identity in 111 out of 112 residues when we compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.

Carboxylmethylation of insulin and glucagon in vitro.

We studied the methyl acceptor capacity of insulin and glucagon in vitro. The levels of carboxylmethylation of pancreatic hormones (dpm x 10(3], when incubated with S-adenosyl-L-(3H-methyl)-methionine as methyl donor and purified protein carboxylmethylase, were: insulin (n = 6) 8.1 +/- 0.2 and 11.1 +/- 1.5 (mean +/- SEM) for 0.25 and 1.0 mg/ml, respectively; glucagon (n = 6) 17.0 +/- 3.2 and 40.2 +/- 2.5 (mean +/- SEM) for 0.5 and 1.0 mg/ml, respectively. On a molar basis, the methyl acceptor capacity was 1.0 dpm/pmol for insulin and 9.5 dpm/pmol for glucagon. Polyacrylamide gel electrophoresis of carboxylmethylated hormones showed a radioactivity (3H-methyl) peak that co-migrated with the corresponding 125I-hormone. Glucagon, but not insulin, seems to be a relatively good substrate for carboxylmethylation.

Human placenta S-adenosylmethionine: protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization.

1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. The Km for S-adenosyl-L-methionine and Ki for S-adenosyl-L-homocysteine were 2.08 x 10(-6) and 5.8 x 10(-7) M, respectively. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.