Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

O-Methylation of flavonoid substrates by a partially purified enzyme from soybean cell suspension cultures.

A methyltransferase, which catalyzes the methylation of luteolin (K_m, 16 μM) using S-adenosyl-l-methionine as the methyl donor, has been purified about 38-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The following 3,4-dihydroxy phenolic compounds were also methylated: luteolin 7-O-glucoside (K_m, 28 μm), quercetin (K_m, 35 μm), eriodictyol (K_m, 75 μm), 5-hydroxyferulic acid (K_m, 227 μm), dihydroquercetin (K_m, 435 μm), and caffeic acid (K_m, 770 μm). Rutin and quercetin 3-O-glucoside were poor substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of p-coumaric acid, m-coumaric acid, ferulic acid, isoferulic acid, sinapic acid, apigenin, or naringenin. While the isoflavones biochanin A and daidzein did not serve as substrates, texasin (6,7-dihydroxy-3′-methoxyisoflavone) was methylated (K_m, 35 μm). The methylation of caffeic acid and quercetin showed a pH optimum of 8.6–8.9. The enzyme required Mg²⁺ ions for maximum activity (approximately 1 mm) and could be totally inhibited by EDTA (10 mm). The K_m for S-adenosyl-l-methionine was 11 μm. S-Adenosyl-l-homocysteine inhibited the methylation of luteolin by S-adenosyl-l-methionine.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Methylation of anthocyanins by cell-free extracts of flower buds of Petunia hybrida

An O-methyltransferase activity which catalyses the methylation of anthocyanins was extracted from flowerbuds of Petunia hybrida. The methyltransferase uses S-adenosyl-l-methionine as methyl donor. Only anthocyanidin 3(p-coumaroyl)rutinosido-5-glucoside was methylated. No methylating activity towards anthocyanidins, anthocyanidin 3-glucosides, anthocyanidin 3-rutinosides, caffeic acid or p-coumaric acid could be detected.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Nadph: Biochanin a oxidoreductase from the fungus Fusarium javanicum

A soluble enzyme which catalyses the NADPH-dependent reduction of the heterocyclic double bond of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxy-isoflavone) yielding the corresponding isoflavanone was isolated from the fungus Fusarium javanicum. The NADPH: biochanin A oxidoreductase was constitutively present in the mycelium with an extractable average activity of 4 pkat/g fresh weight. The enzyme was purified ca 4500 fold to apparent homogeneity. The native enzyme had M_r, of ca 87 000 and consisted of two identical subunits of M_r, 43 000. The enzyme reaction showed a pH-optimum at pH 7.5 and a temperature optimum between 30 and 35°. The apparent K_m values were 43 μM for biochanin A and 190 μM for NADPH with a maximum velocity of 4 mkat/kg protein. The enzyme exhibited a remarkable substrate specificity for biochanin A.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

Inhibitors of two enzymes which metabolize cytokinins

Compounds which inhibit the natural metabolic inactivation of cytokinins are of considerable physiological significance. In this study, inhibitors have been found for two enzymes which form glucose and alanine conjugates of cytokinin bases, namely, cytokinin 7-glucosyltransferase and β-(9-cytokinin)alanine synthase. The most effective inhibitors found for the former enzyme were the cytokinin analogues 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine, which acted competitively (K_i, 22 μM), and the diaminopurine, 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine (K_i, 3.3 μM). However these compounds were ineffective as inhibitors of the cytokinin-alanine synthase which was inhibited competitively by IAA (K_i 70 μM) and related compounds, especially 5,7-dichloro-IAA (K_i 0.4 μM). Certain urea derivatives were moderately effective inhibitors of the enzymes (K_ica 100μM).     

S-adenosylhomocysteine hydrolases as the primary target enzymes in androgen regulation of methylation complexes

tRNA methylation complexes consisting of S-adenosylmethionine (AdoMet) synthetase, tRNA methylases, and S-adenosylhomocysteine (AdoHcy) hydrolase have been prepared from rat Novikoff hepatoma cells. The existence of the ternary enzyme complex is supported by dissociation and reconstitution of the ternany tRNA methylation complexes. In rat prostate and testis, two isozymes each for AdoMet synthetase and AdoHcy hydrolase are detected. The K_m (methionine) values for the two AdoMet synthetases are 3.1 and 23.7 μm and the K_m (adenosine) values for the two AdoHcy hydrolases are 0.33 and 1.8 μm. Correspondingly, two groups of methylation complexes are detectable, sedimenting in a sucrose gradient as 7 S and 8 S. The 7 S complexes are composed of AdoMet synthetase and AdoHcy hydrolase with the higher K_m values, and the 8 S complexes are composed of the respective isozymes with the lower K_m values. tRNA methylation complexes belong to the 8 S group. In hormone-depleted rat prostates and testes following hypophysectomy, the specific activities of AdoMet synthetases, tRNA methylases, and AdoHcy hydrolases are decreased severely, but are restored promptly after administration of testosterone. Thus, methylation enzymes are responsive to the regulation by steroid hormone. AdoHcy hydrolases from hormone-depleted tissues are unstable, and ternary tRNA methylation complexes are easily dissociable into individual activities. The stability of AdoHcy hydrolases is markedly improved by testosterone, and the integrity of ternary tRNA methylation complexes is maintained in the presence of testosterone. These results suggest that AdoHcy hydrolases are the primary target enzymes in adrogen regulation of methylation complexes.     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

Membrane-bound O-methyltransferase of douglas-fir callus

A lignin-specific O-methyltransferase (OMT) was localized in the cell wall fraction of Douglas-fir needle callus homogenates. The OMT was released from wall-associated membrane by digitonin and partially purified by salt fractionation. Further purification proved to be unfeasible, due to the high tannin content of the callus. The K_m values of the partially purified OMT for caffeic acid and S-adenosylmethionine (SAM) were 250 and 8.0.μM, respectively. Substrate inhibition as well as inhibition by S-adenosylhomocysteine (SAH) was observed. Coupled with low levels of caffeic acid found in the callus, 65,μM at maximum with a mean of 11.5μM throughout a subculture period, the properties of this OMT should account in large part for the high tannin and low lignin content characteristic of this cultured tissue.     

Polyamine oxidase of millet shoots

Polyamine oxidase was purified ca 168-fold from the acetone powder extract of millet shoots. The light yellow enzyme had maximum absorption at 278,380 and 460 nm. The absorption at 380 and 460 nm was decreased by the addition of spermidine. The enzyme (M, ca 80 000) showed a high specificity for spermine and spermidine (K_ms 6 × 10⁻⁵ M and 5 × 10⁻⁷ M respectively). The enzyme was inhibited by quinacrine and acriflavine.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

Properties of hydroxycinnamate: CoA ligase from stems of Salix babylonica cultured in vitro

Hydroxycinnamate: CoA ligase was extracted from stems of in vitro willow cultures and characterized. One peak of activity was obtained after column chromatography on Sephadex G 100 or DEAE Sephacel. p-Coumaric acid gave the highest V_max among the cinnamates examined. The K_mvalues for p-coumaric, caffeic and ferulic acid were 31.0, 4.7 and 46 μM, respectively. The MW of the CoA ligase was 57 000 and the pH optimum was 7.0. The characteristics of the enzyme correspond to its physiological role in lignin biosynthesis.     

Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl-l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH₄)₂SO₄ precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.     

Chloroplast cysteine synthases of Trifolium repens and Pisum sativum

About 68–86% of the cysteine synthase activity in leaf tissue of white clover (Trifolium repens) and peas (Pisum sativum cultivar Massey Gem) was associated with chloroplasts. The enzymes from white clover and peas were purified ca 66 and 12-fold respectively. For clover, the K_m values determined by calorimetric and S^2? ion electrode methods were: S^2? 0.51 and 0.13 mM; O-acetylserine (OAS), 3.5 and 2.O mM respectively. The analogous values for the pea enzyme were: S^2?, 0.24 and 0.06 mM; OAS, 3.1 and 0.24 mM. Both enzymes were inhibited by cystathionine and cysteine. Pretreatment with cysteine inactivated the enzyme, but addition of pyridoxal phosphate caused partial reactivation. Isolated pea chloroplasts (70–75 % intact) catalysed OAS-dependent assimilation of sulphide at a mean rate of 88 μmol/mg Chl/hr. About 85 % of the OAS-dependent sulphide assimilated was recovered as cysteine. The rates were unaffected by light and 2 μM DCMU. Sonicating the chloroplasts enhanced the rate by 1.3–2 fold. Cysteine synthase activity was associated with the chloroplast stroma. Similar results were obtained for clover chloroplasts except that both the intactness and the rates were lower.     

The effects of tetrahydroisoquinolinecarboxylic acids on tyrosine 3-monooxygenase

The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor (K_i = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor (K_i = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid (K_i = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid (K_i = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro, decreases in the K_i of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the K_i of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC₅₀ = 100 μm) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo.