High yield production of S-adenosyl-l-homocysteine with microbial cells as the catalyst

A simple and efficient enzymatic process for the production of S-adenosyl-l-homocysteine through catalysis by bacterial S-adenosylhomocysteine hydrolase (EC 3.3.1.1) is described. Washed cells of several actinomycetes, and Alcaligenes and Pseudomonas strains were found to be favorable sources of the enzyme. The reaction is carried out in potassium phosphate, pH 8.0, containing adenosine and l-homocysteine as the substrates, and washed cells of Alcaligenes faecalis AKU 101, which contain a high level of the enzyme, as the catalyst. With concentrations of adenosine and l-homocysteine of up to 200 mM, the molar conversion to S-adenosyl-l-homocysteine after 4–21 h at 37°C was nearly 100%. The maximum yield of 199 mM (76.5 mg ml⁻¹) was attained with a reaction mixture containing 300 mM substrates. S-Adenosyl-l-homocysteine of a high purity was easily isolated from the reaction mixture by simple gel-filtration on Sephadex G-10 with good recovery.     

A sensitive radiochemical enzyme assay for S-adenosyl-l-homocysteine and l-homocysteine

The reversibility of the S-adenosylhomocysteine hydrolase reaction allows both nonradiolabeled S-adenosyl-l-homocysteine and l-homocysteine to serve as l-homocysteine donors for the synthesis of radiolabeled S-adenosyl-l-homocysteine from radiolabeled adenosine. Using high specific activity, radiolabeled adenosine, and high-performance liquid chromatography to separate products and reactants, as little as 0.1 pmol of l-homocysteine donor can be detected by its ability to be converted to radiolabeled S-adenosyl-l-homocysteine. Reduction of samples with dithiothreitol during the enzymatic reaction allows l-homocystine and mixed disulfides of l-homocysteine to be assayed as well. S-Adenosyl-l-homocysteine can be distinguished from other l-homocysteine donors by assaying samples before and after degradation of the former with the nonspecific adenosine deaminase from Aspergillus oryzae. A less sensitive version of the assay employs thin-layer chromatography in place of high-performance liquid chromatography, and is useful for the assay of l-homocysteine and its disulfides in quantities of 25 pmol or more.     

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Adenosine metabolism: Modification by S-adenosylhomocysteine and 5′-methylthioadenosine

To elucidate potential toxic properties of S-adenosylhomocysteine and 5′-methylthioadenosine, we have examined the inhibitory properties of these compounds upon enzymes involved with adenosine metabolism. S-Adenosylhomocysteine, but not S-adenosylmethionine, was a noncompetitive inhibitor of adenosine kinase with K_i values ranging from 100 to 400 μm. Methylthioadenosine competitively inhibited adenosine kinase with variable adenosine below 1 μm with a K_i of 120 μm, increased adenosine kinase activity when the adenosine concentration exceeded 2 μm, and did not appear to be a substrate for adenosine kinase. Methylthioadenosine inactivated S-adenosylhomocysteine hydrolase from erythrocytes, B-lymphoblasts, and T-lymphoblasts with K_i values ranging from 65 to 117 μm and “k₂” from 0.30 to 0.55 min^?1. Adenosine deaminase was not inhibited by 5′-methylthioadenosine up to 1000 μm. To clarify how 5′-methylthioadenosine might accumulate, 5′-methylthioadenosine phosphorylase was evaluated. This enzyme was not blocked by up to 500 μm adenosine, deoxyadenosine, S-adenosylhomocysteine, or S-adenosylmethionine and was not decreased in erythrocytes from patients with adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, or hypogammaglobulinemia. These observations suggest that the inhibitory properties of 5′-methylthioadenosine upon adenosine kinase and S-adenosylhomocysteine hydrolase may contribute to the toxicity of the exogenously added compound. The toxicity resulting from S-adenosylhomocysteine accumulation intracellularly may be related to adenosine kinase inhibition in addition to disruption of transmethylation reactions.     

Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer’s disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.     

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S-adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S-adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S-adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S-adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S-adenosylhomocysteine inhibit some transmethylation reactions.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

(1) The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. (2) A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinteic parameters of S-adenosylhomocysteine hydrolase. (3) During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 μM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 μM) caused an increased of 0.37 and 4.17 nmol SAdony/soc per g wet weight during normaxia and ischemia, respectively. (4) The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 115 nmol/g; P < 0.05). Purine release was increased 4-fold during ischemia. (5) The K_m for hydrolysis of SAdoHcy was about 12 μM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 μM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. (6) From the combined in vitro and perfusion studies, we comclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.     

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

S-Adenosylhomocysteine hydrolase (SAHH) is an NAD⁺-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys⁴⁰¹ and Lys⁴⁰⁸ but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys⁴⁰¹ and Lys⁴⁰⁸ and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD⁺ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.     

Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml⁻¹ OD⁻¹ compared with the signal peptide of V. cholerae CtxB (52 mU ml⁻¹ OD⁻¹). This positive effect was confirmed with a β-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher β-glucosidase activity (181 mU ml⁻¹ OD⁻¹) than with the CtxB signal peptide (42 mU ml⁻¹ OD⁻¹). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.     

Purification and properties of S-adenosyl-L-homocysteine hydrolase from leaves of spinach beet.

S-Adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and purified 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine and its synthesis from these compounds. The equilibrium constant for the reaction is 1.8 × 10^?6 in relation to hydrolysis. The enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The K_m for S-adenosylhomocysteine was 41 μm in the hydrolysis reaction and for adenosine, dl-homocysteine, and l-homocysteine it was 13 μm, 2.2 mm, and 1.2 mm, respectively.The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the K_m values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using S-adenosylmethionine as methyl donor.     

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5′ analogues

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.     

Methionine metabolism in mammals: S-adenosylhomocysteine hydrolase in rat intestinal mucosa.

Extracts of rat small intestinal mucosa can catalyze the synthesis of adenosylhomocysteine from homocysteine and adenosine. In order to demonstrate this reaction, adenosine deaminase, which competes for the substrate, must be inhibited or removed by purification. We have also demonstrated catabolism of adenosylhomocysteine during incubation with intestinal extracts. The immediate reaction products are adenosine and homocysteine. Thus, small intestinal mucosa contains both reactivities characteristic of S-adenosylhomocysteine hydrolase. This enzyme has been found in all mammalian tissues that have been studied.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.     

A new structural class of S-adenosylhomocysteine hydrolase inhibitors

Effective inhibitors of S-adenosylhomocysteine hydrolase hold promise towards becoming useful therapeutic agents. Since most efforts have focused on the development of nucleoside analog inhibitors, issues regarding bioavailability and selectivity have been major challenges. Considering the marine sponge metabolite ilimaquinone was found to be a competitive inhibitor of S-adenosylhomocysteine hydrolase, new opportunities for developing selective new inhibitors of this enzyme have become available. Based on the activities of various hybrid analogs, SAR studies, pharmacophore modeling, and computer docking studies have lead to a predictive understanding of ilimaquinone’s S-adenosylhomocysteine hydrolase inhibitory activities. These studies have allowed for the design and preparation of simplified structural variants possessing new furanoside bioisosteres with 100-fold greater inhibitory activities than that of the natural product.     

S-Adenosylhomocysteine Hydrolase Deficiency in Cysteine Auxotrophs of Tetrahymena thermophila1

The biochemical lesion in two cysteine auxotrophs of Tetrahymena thermophila has been established as a defect in S-adenosylhomocysteine hydrolase, an enzyme of the transsulfuration pathway. As a result, these mutants require cysteine (or cystathionine or homocysteine) for growth in a denned medium. Cell-free extracts of the mutants contained < 5% of the level of the enzyme seen in the wild type. One of the mutant strains accumulated intracellular levels of S-adenosylhomocysteine as high as 1380 üM, a level 200 times normal. When both mutant strains were maintained in defined medium without cysteine, growth occurred after a long lag; this phenomenon was termed “adaptation.” Adaptation was a) reversed by passage through rich medium, b) was not a recovery of S-adenosylhomocysteine hydrolase, and c) was probably linked to induction of an alternate pathway for cysteine biosynthesis, involving a lysosomal S-adenosylhomocysteine nucleosidase activity.     

Enzymatic synthesis of <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine: immobilization of recombinant <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine hydrolase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> (ATCC 13032)

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD⁺ release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD⁺ to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.