S-adenosyl-L-methionine Concentrations Research Articles

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM)is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, anSAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15–33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g−1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L−1L-met and 0.60 g L−1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L−1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L−1 to 10.1 g L−1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl-l-methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols.

Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae.

S-Adenosyl-l-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l-methionine is generally used, the low-cost dl-methionine is more suitable as the substrate for industrial production of SAM. However, d-methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl-methionine, intracellular conversion of d-methionine to l-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d-amino acid-N-acetyltransferase, d-methionine was accumulated in vivo and no N-acetyl-d-methionine production was observed. Further, codon-optimized d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l-phenylalanine dehydrogenase gene (l-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d-methionine to l-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d-methionine. Using the recently developed CRISPR tools, the DAAO and l-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl-methionine.

Rapid and Quantitative Determination of S-Adenosyl-L-Methionine in the Fermentation Process by Surface-Enhanced Raman Scattering.

Concentrations of S-Adenosyl-L-Methionine (SAM) in aqueous solution and fermentation liquids were quantitatively determined by surface-enhanced Raman scattering (SERS) and verified by high-pressure liquid chromatography (HPLC). The Ag nanoparticle/silicon nanowire array substrate was fabricated and employed as an active SERS substrate to indirectly measure the SAM concentration. The linear relationship between the integrated intensity of peak centered at ~2920 cm−1 in SERS spectra and the SAM concentration was established, and the limit of detections of SAM concentrations was analyzed to be ~0.1 g/L. The concentration of SAM in real solution could be predicted by the linear relationship and verified by the HPLC detection method. The relative deviations (δ) of the predicted SAM concentration are less than 13% and the correlation coefficient is 0.9998. Rolling-Circle Filter was utilized to subtract fluorescence background and the optimal results were obtained when the radius of the analyzing circle is 650 cm−1.

13C-metabolic flux analysis in S-adenosyl-l-methionine production by Saccharomyces cerevisiae

S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

Synthesis of S-adenosyl-L-methionine in Escherichia coli

Abstract S-adenosyl-L-methionine (SAM) is an important physiological metabolite in vivo and may be useful in medicines. SAM is produced from L-methionine and ATP catalyzed by S-adenosyl-L-methionine synthetase (SAMS) in vivo. In this study, the gene encoding SAMS was cloned and a genetically engineered Escherichia coli (E. coli) BL21(pET-28a-SAMS) was constructed. The recombinant SAMS with a molecular mass of approximately 46 kDa was expressed by inducing the engineered E. coli using isopropyl-s-D-1-thiogalactopyranoside (IPTG) as an inducer. To produce SAM using a low-cost, nontoxic and highperformance expression system, lactose was used as a substitute for IPTG to induce BL21(pET-28a-SAMS). By optimizing the expression conditions, the concentration of SAM produced by the engineered E. coli was 48 mg/L in the culture medium supernatant. To increase the concentration of SAM produced, a coupled system was constructed consisting of E. coli BL21(pET-28a-SAMS) and Saccharomyces cerevisiae (S. cerevisiae) JM-310. In this coupled system, ATP generated from S. cerevisiae was provided to E. coli for producing a higher concentration of SAM. The SAM concentration in the coupled system reached 1.7 g/L. SAM was purified by a weak acid cationic exchange resin D113, and a simple and economical purification procedure for SAM isolation was achieved. SAM was confirmed by High Performance Liquid Chromatography-tandem Mass Spectrometry analysis. Our study provides a feasible and convenient approach to produce SAM.

Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase.

Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA•). In the LS reaction, two equivalents of 5′-dA• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N6-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]0 clusters.

P17 Modulatory effect of S-adenyl-methionine, an allosteric activator of cystathionine-beta-synthase on the proliferation and bioenergetics of colon cancer cells: Mechanism of action

Previous studies reported that S-adenosyl- l -methionine (SAM), an allosteric activator of cystathionine-beta-synthase (CBS) increases the production of hydrogen sulfide (H2S) in isolated enzyme preparations. Given the multiple pharmacological effects of SAM, the goal of our study was to probe the specific role of CBS in the effects of SAM in HCT116 colon cancer cells. SAM (0.1 μM–3 mM) concentration-dependently increased CBS-mediated H2S production. The effect of SAM on cell proliferation (assessed real time for up to 24 h by xCELLigence) was time-dependent. SAM (0.1–3 mM) enhanced HCT116 cell proliferation at all concentrations tested in the first 12 h following treatment, but induced cell death/inhibition of proliferation thereafter (12–24 h). The short-term stimulatory effects of SAM were attenuated by stable CBS silencing. In contrast, the anti-proliferative effects of SAM were maintained in cells with CBS silencing. Short-term exposure of HCT116 cells to SAM (0.1–1 mM, 1 h) concentration-dependently increased the oxygen consumption and bioenergetic function of HCT116 cells, while longer-term exposures resulted in a suppression of these parameters at all concentrations of SAM tested. While the stimulatory effect of SAM on proliferation and bioenergetics were attenuated in cells with stable CBS silencing, the inhibitory effects of SAM were largely unaffected by it. Thus, CBS in colon cancer cells produces H2S in a regulated fashion. H2S, when produced at low-to-intermediate levels, serves as an endogenous cancer cell growth and bioenergetic factor. While the short-term inhibition of cell proliferation by high concentrations of SAM may be partially related to “over-stimulation” of CBS, the long-term inhibitory effects of SAM on cell proliferation and bioenergetics are mainly CBS-independent.

Effect of S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro

Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-l-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1–3mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1–1mM SAM increased HCT116 proliferation between 0 and 12h, while the highest SAM concentration (3mM) inhibited proliferation. Over a longer time period (12–24h), only the lowest concentration of SAM used (0.1mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1–1mM, while 3mM was inhibitory. Longer-term (72h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.

P03 Effect of S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro

Several series of in vitro and in vivo data presented at the current Meeting support the conclusion that colon cancer cells selectively overexpress CBS, which produces H2S, to maintain cellular bioenergetics, thereby supporting tumor growth and to promote angiogenesis and vasorelaxation. These studies, taken together, identify CBS-derived H2S as a tumor promoting factor and a potential future anticancer drug target. The purpose of the current study was to investigate the effect of S-adenosyl-l-methionine (SAM) on the proliferation and bioenergetics of the colon cancer cell line HCT116 in vitro. The non-transformed, the non-tumorigenic colon epithelial cell line NCM356, derived from the normal margin of a rectal cancer specimen, was used as a control. HCT116 cells were cultured in McCoy’s 5A medium, while NCM356 cells were cultured in DMEM supplemented with 10% fetal bovine serum. For assessment of cell proliferation, the xCELLigence system (Roche) was used. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer (Seahorse) was used. Oxygen consumption rate (OCR) after oligomycin (1.5 μg/ml) was used to assess ATP production rate and OCR after FCCP (0.5 μM) to assess maximal mitochondrial respiratory capacity. 2-deoxyglucose (100 mM) was used to estimate cellular glycolytic dependency and antimycin A (2 μg/m) and rotenone (2 μM) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial activity. Addition of SAM (0.1–1 mM) to HCT116 cells (which show a high expression of CBS) induced a concentration-dependent increase in cell proliferation between 0–50 h, while higher concentrations of SAM (3 mM) inhibited cell proliferation. However, at time periods between 50–100 h, the cells treated with intermediary concentrations of SAM (0.3–1 mM) showed a suppression of cell proliferation compared to the vehicle control; the proliferation-enhancing effect of SAM was only maintained throughout the entire experimental period with the 0.1 mM concentration of SAM. In contrast to HCT116 cells, NCM356 cells (which have only a low-level expression of CBS) were proliferating at a substantially slower rate, and SAM failed to stimulate their proliferation. Extracellular Flux Analysis of the effect of acute exposure of HCT116 cells to SAM (0.1–1 mM) induced a concentration-dependent increase in their oxygen consumption and bioenergetic function, while in NCM356 cells SAM failed to enhance oxygen consumption and cellular bioenergetics. Longer-term exposure of HCT116 cells to both low concentrations of SAM (0.1 mM) and high concentrations of SAM (1 mM) suppressed cellular bioenergetic responses. Studies presented at the current meeting demonstrate that the H2S-producing enzyme CBS is selectively overexpressed in human colorectal cancer cells, and significantly contributes to their proliferation, migration and invasion in vitro. The present data, demonstrating that short-term exposure of SAM to HCT116 cells exerts proliferative and positive bioenergetic effects that are in line with the effects of exogenously administered H2S in the same experimental system further support the view that CBS in colon cancer cells produces H2S in a regulated fashion, and that this H2S serves as an endogenous cancer cell proliferation and bioenergetic factor. Higher local concentrations of H2S are known to be antiproliferative, at least in part due to their inhibitory on mitochondrial function. Therefore, the longer-term inhibition of cell proliferation and bioenergetic function noted with SAM in the present experiments may either be attributed to the adverse autocrine effects of high concentrations of H2S, produced when CBS is ‘over-activated’ by SAM, or, alternatively, may be related to additional, CBS-independent pharmacological effects of SAM.

Influence of harvest time and 1-MCP application on postharvest ripening and ethylene biosynthesis of ‘Jonagold’ apple

Changes in ethylene biosynthesis, respiration and firmness were studied during on-tree ripening, controlled atmosphere (CA) storage and shelf-life of ‘Jonagold’ apples. Particular attention was paid to the influence of harvest time on subsequent ripening during CA storage and shelf-life and on the effect of treatment with the ethylene inhibitor 1-MCP. The influence of harvest time was most prominent on firmness, with late harvested apples being significantly softer throughout the whole postharvest life. Respiration rate was mainly influenced by the surrounding temperature and air conditions and to a lesser extent by the harvest time. Ethylene production rate depended on harvest time straight after harvest and during storage but this effect disappeared upon movement from storage to shelf-life. When apples were treated with 1-MCP, ethylene biosynthesis was almost completely suppressed throughout the whole postharvest life, with exception of the late harvested apples which regained some ethylene forming capacity after two weeks of shelf-life. A detailed overview of ethylene biosynthesis including the often neglected metabolite S-adenosyl-l-methionine (SAM) was generated. The changes in SAM concentration could not directly be linked to the changes in ethylene production rate as such and SAM was not a limiting factor in apple ethylene biosynthesis during ripening. In vitro 1-aminocyclopropane-1-carboxylate synthase (ACS) and oxidase (ACO) activities were differently affected by CA storage conditions with ACS being the most dominant controlling factor in the biosynthesis of ethylene.

Kinetic Mechanism of Protein Arginine Methyltransferase 6 (PRMT6)

The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the mono- and dimethylation of arginine residues in a variety of proteins. Although these enzymes play important roles in a variety of cellular processes, aberrant PRMT activity is associated with several disease states, including heart disease and cancer. In an effort to guide the development of inhibitors targeting individual PRMTs, we initiated studies to characterize the molecular mechanisms of PRMT catalysis. Herein, we report studies on the kinetic mechanism of PRMT6. Initial velocity, product inhibition, and dead-end analog inhibition studies with the AcH4-21 and R1 peptides, as well as their monomethylated versions, indicate, in contrast to a previous report, that PRMT6 utilizes a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes.

Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-l-methionine reveals regulation of TPMT*1 and *3C allozymes

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-l-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n=96) compared to the low SAM level group (n=19; P<0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction of l-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.

S-Adenosyl-L-Methionine Induced RyR2 Subconductance: Evidence for an Allosteric Mechanism

We previously reported (Kampfer & Balog Biochem 49:7600, 2010) that S-adenosyl-l-methionine (SAM) can act as a RyR2 channel regulatory ligand in a manner independent from its recognized role as a biological methyl group donor. Channel activation appeared to arise from the interaction of SAM with a RyR2 adenine nucleotide binding site. In addition to its ability to activate RyR2, single channel recordings revealed distinct effects of SAM on RyR2 conductance, which we explored here in greater detail. The effects of SAM on native RyR2 channel conductance in symmetric cesium methanesulfonate were dependent on SAM concentration and holding potential. At negative potentials, cis SAM induced a single, clearly resolved subconductance state (∼2/3 full conductance). The proportion of SAM induced subconductance openings, as a proportion of all openings (Psub/Po), increased with decreasing negative potential. Kinetic analysis revealed that changes in the SAM off rate accounted for the voltage dependence of the transitions between the full open and SAM induced subconductance state. In contrast, at positive potentials SAM caused a striking reduction in channel openings with no distinct effect on channel conductance. Inconsistent with a simple pore block mechanism was the finding that the prevalence of the subconductance state was unaffected by varying the cesium concentration gradient across the bilayer. Furthermore, ATP but not 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials, suggesting ATP competition with SAM for a common binding site. We interpret these findings in light of an allosteric mechanism whereby SAM interacts with a RyR2 adenine nucleotide binding site. SAM binds to and stabilizes a channel conformation of reduced conductance and we propose the voltage dependence of the SAM-induced subconductance state arises from a voltage-driven alteration in the affinity of the SAM binding site.

Variable Sequences outside the Sam-Binding Core Critically Influence the Conformational Dynamics of the SAM-III/SMK Box Riboswitch

The S MK box (SAM-III) translational riboswitches were identified in S-adenosyl- l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S MK box riboswitch. In this study, we combined selective 2′-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S MK box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S MK variants display a “constitutively OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2′-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S MK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S MK box RNA follows a two-step folding/unfolding process.

MODELING OF ETHYLENE BIOSYNTHESIS DURING RIPENING AND CA STORAGE OF 'JONAGOLD' APPLES

Simulation models can make the determination of optimal storage conditions for new cultivars of Malus domestica less expensive and less time consuming. On top of this, modeling creates the opportunity to apply batch dependent conditions. Two experiments were set up to develop a model to describe ethylene production during the ripening and storage of 'Jonagold' apples. Fruit were harvested at different dates around the optimal harvest date to obtain different ethylene production rates at different ripeness stages. Apples harvested at the three most extreme harvest dates were used for the storage experiment (CA conditions, 1% O-2, 3% CO2) and either treated or not treated with Smartfresh (TM), a commercially applied ethylene inhibitor. The ethylene production rate was determined by gas chromatography and S-adenosyl-L-methionine (SAM) concentrations were measured with capillary electrophoresis. During fruit ripening and during the storage of untreated fruit, an increase in ethylene production rate was noticed. SAM concentrations on the other hand were not influenced significantly during the 4 weeks period around optimal harvest time.

S-Adenosyl-l-methionine Regulation of the Cardiac Ryanodine Receptor Involves Multiple Mechanisms

Cardiac contraction is triggered by the release of Ca(2+) via the ryanodine receptor (RyR2), a sarcoplasmic reticulum (SR) resident ion channel. RyR2 channel activity is modulated through ligand binding and posttranslational regulatory mechanisms. S-Adenosyl-l-methionine (SAM), the primary methyl group donor for enzyme-mediated methylation of proteins and other biological targets, activates RyR2 via an unknown mechanism. Here we show that the SAM-induced increase in cardiac SR (CSR) vesicle [(3)H]ryanodine binding is unaffected by methyltransferase inhibitors and immunoprecipitation of RyR2 from S-adenosyl-l-[methyl-(3)H]methionine ([(3)H]SAM) pretreated CSR indicates that RyR2 is not a target of SAM-mediated protein methylation. Because SAM contains an adenosine moiety and RyR2 is activated by ATP, we investigated whether SAM exerts its effects through the adenine nucleotide binding sites on the RyR2 channel. In support of this hypothesis, the SAM and ATP concentration dependence of CSR vesicle [(3)H]ryanodine binding virtually overlaps. Furthermore, ryanodine binding assays show that SAM competes with adenine nucleotide activation of RyR2, and the effects of SAM on mean channel open and closed times follow similar trends as those observed for ATP. Interestingly, SAM but not ATP activation of RyR2 was associated with a marked increase in the percent of channel openings to a subconductance level approximately 60% of the maximal single channel conductance. This work highlights the complexity underlying SAM regulation of RyR2 and suggests ligand binding is among the multiple mechanisms responsible for SAM regulation of RyR2.

DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl- l-methionine production in Pichia pastoris

S-Adenosyl- l-methionine (SAM) is an important molecule for normal cell function and survival. To enhance methionine adenosyltransferase (MAT, EC2.5.1.6) activity and thus SAM production in recombinant Pichia pastoris, MAT genes from Escherichia coli, Saccharmyces cerevisiae, and Streptomyces spectabilis were recombined by DNA shuffling. The shuffled genes were transformed into P. pastoris GS115 to construct recombinant strains and screened for high MAT activity and enhanced SAM production mutants. In the two best recombinant strains, the MAT activities were respectively 201% and 65% higher than the recombinant strains containing the starting MAT genes, and the SAM concentration increased by 103% and 65% respectively. The analysis on the deduced sequences of five representative MAT variants showed that the K18R mutation probably resulted in the increased activity of the best MAT, which may provide new insights into structure-enzyme activity relationship of MAT and might shed light on the further rational evolution of MAT. Finally, a 6.14 g l −1 of SAM production was reached in a 500 l bioreactor with the best recombinant strain GS115/DS56, which showed a favorable foreground in industrial scale production of SAM.

SAM Regulation of RyR2

RyR2 channel activity is subject to allosteric and posttranslational regulation. S-adenosyl-L-methionine (SAM), the primary methyl group donor for enzyme-mediated methylation of proteins and other biological targets, activates RyR2 via an unknown mechanism. To determine if activation of RyR2 by SAM requires methyltransferase activity, cardiac sarcoplasmic reticulum (CSR) vesicle [3H]ryanodine binding was performed in media containing 150 mM KCl, 20 mM PIPES pH 7.0, 3 μM Ca2+, 1 mM SAM, and 0–1 mM sinefungin, a competitive methyltransferase inhibitor. Sinefungin did not alter CSR vesicle [3H]ryanodine binding, and the SAM-induced increase in [3H]ryanodine binding was not altered by sinefungin. To investigate further whether activation of RyR2 by SAM involves RyR2 methylation, RyR2 was immunoprecipitated from CSR vesicles pretreated for 30 min at 37°C with 3 μCi; 285 nM [3H]SAM plus or minus 500 fold excess cold SAM or 1 mM SAH, an inhibitor of methyltransferase activity, followed by centrifugation through sucrose. Radioactivity incorporated into pretreated CSR and immunoprecipitated RyR2 was determined by liquid scintillation counting. Although the amount of radioactivity incorporated into [3H]SAM pretreated CSR vesicles was reduced from 6.54 to 1.86 and 1.69 pmol [3H]SAM/mg protein in the presence of SAH and excess cold SAM respectively, the amount of radioactivity recovered by immunoprecipitation with anti-RyR was not increased over control (immunoprecipitation without RyR specific antibody). Because SAM contains an adenosine moiety and may activate RyR2 via interaction with the channel's adenine nucleotide binding site(s), the affects of SAM and ATP on RyR2 activity were compared. The SAM and ATP concentration dependence of CSR vesicle [3H]ryanodine binding virtually overlapped with no differences at any concentration tested. This work suggests SAM does not methylate RyR2 and the similarities between ATP- and SAM-induced RyR2 activation support allosteric regulation of RyR2 by SAM.