Genomic characterization of the S-adenosylmethionine decarboxylase genes from soybean

A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5 leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5 leader sequence, but no intron in the 3-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.Communicated by H. F. Linskens     

S-adenosylmethionine decarboxylase from baker''s yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.     

Crenarchaeal arginine decarboxylase evolved from an S-adenosylmethionine decarboxylase enzyme

The crenarchaeon Sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. However, genome sequences from S. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5'-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. Instead they have two paralogs of the S-adenosylmethionine decarboxylase (AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both thermostable enzymes self-cleave at conserved serine residues to form amino-terminal beta-domains and carboxyl-terminal alpha-domains with reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine decarboxylation more efficiently than previously studied pyruvoyl enzymes. alpha-Difluoromethylarginine significantly reduced the ArgDC activity of purified enzyme, and treating growing S. solfataricus cells with this inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the beta-subunit of SSO0536 and the alpha-subunit of SSO0585 had ArgDC activity, implicating residues responsible for substrate specificity in the amino-terminal domain. This crenarchaeal ArgDC is the first example of alternative substrate specificity in the AdoMetDC family. ArgDC activity has evolved through convergent evolution at least five times, demonstrating the utility of this enzyme and the plasticity of amino acid decarboxylases.     

Comparison of inhibitors of S-adenosylmethionine decarboxylase from different species.

S-Adenosyl-L-methionine decarboxylases were purified from rat ventral prostate, yeast (Saccharomyces cerevisiae), slime mould (Physarum polycephalum) and bacteria (Escherichia coli) and tested for inhibition by a variety of nucleosides related to S-adenosylmethionine and by methyl- and ethyl-glyoxal bis(guanylhydrazone). Although the enzymes from these different sources are markedly different with respect to activation by cations, the inhibition by nucleosides was quite similar. Very little inhibition was seen when analogues of S-adenosylmethionine with a different base were tested or when the ribose ring was opened or the positive charge on the sulphur atom was not present. Some derivatives in which the amino acid portion of the molecule was altered were more potent inhibitors, but again there was little difference between the enzymes from different sources. 5'-(Dimethylsulphonio)-5'-deoxyadenosine and S-adenosyl-3-methylthiopropylamine were the most inhibitory substances and had similar Ki values, suggesting that the aminopropyl group does not contribute significantly to the binding. All of the S-adenosylmethionine decarboxylases were strongly competitively inhibited by methylglyoxal bis(guanylhydrazone) and even more powerfully by its ethyl analogue, although the putrescine-activated enzymes from prostate and yeast were more sensitive than the bacterial and slime-mould enzymes. All of the S-adenosylmethionine decarboxylases tested bound to a column of methylglyoxal bis(guanylhydrazone) linked to Sepharose and were not eluted by 0.5 M-NaCl, but could be released by 1 mM concentrations of the drug, providing a rapid and efficient method for their purification.     

Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells.

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5'-deoxy-5'-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.     

Feedback regulation of S-adenosylmethionine decarboxylase synthesis.

Treatment of Ehrlich ascites-tumour cells with 1-amino-oxy-3-aminopropane (AOAP), a potent inhibitor of ornithine decarboxylase, resulted in a marked decrease in cellular contents of putrescine and spermidine, concomitant with an arrest of cell growth. The activity of S-adenosylmethionine decarboxylase (AdoMetDC) was greatly increased in cells treated with AOAP. This increase in AdoMetDC activity was shown to be, at least partly, caused by enhanced synthesis of the enzyme, which most likely was induced by the change in cellular polyamine content.     

Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei

Trypanosoma brucei brucei contained a S-adenosyl-L-methionine decarboxylase (AdoMetDC) strongly activated by putrescine. The enzyme was also activated to a lesser extent by cadaverine and 1,3-diaminopropane. Spermidine and spermine had no effect on basal activity of the enzyme. However, they interfered with putrescine activation of trypanosomal AdoMetDC. The trypanosomal enzyme could not be precipitated with antiserum against human AdoMetDC. The trypanosomal AdoMetDC enzyme subunit was labeled by reaction with ³⁵S-decarboxylated AdoMet in the presence of NaCNBH₄, and found to have a molecular weight of 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit was readily degraded on storage to a form with a molecular weight of 26 kDa. The specificity of labeling of AdoMetDC by this procedure was confirmed by the prevention of ³⁵S-decarboxylated S-adenosylmethionine (AdoMet) binding in the presence of specific AdoMetDC inhibitors [either methylglyoxal bis(guanylhydrazone (MGBG), a reversible inhibitor, or 5-deoxy-5-[(2-hydrazinoethyl)methylamino]adenosine (MHZEA), an irreversible inactivator]. As compared to human AdoMetDC, the trypanosomal enzyme showed weaker binding to a column of MGBG-Sepharose and also was significantly less sensitive to inhibition by MGBG and its congener ethylglyoxal bis(guanylhydrazone) (EGBG). Thus, the trypanosomal AdoMetDC differs significantly from its mammalian and bacterial counterparts and may therefore be exploited as a specific target for chemotherapy of trypanosomiasis.     

S-adenosylmethionine decarboxylase and spermidine synthase from chinese cabbage

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virus-infected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO₂ and of decarboxylated S-adenosylmethionine (dSAM). The K_m for SAM is 38 micromolar. The reaction is not stimulated by Mg⁺⁺ or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5.1.16), present in leaf or protoplast extracts in many fold excess over SAM decarboxylase, has been purified approximately 1900-fold in steps involving ammonium sulfate precipitation, affinity chromatography, and gel filtration on Sephacryl S-300. Standardization of the Sephacryl column by proteins of known molecular weight yielded values of 35,000 and 81,000 for the decarboxylase and synthase, respectively.     

Nucleotide sequence of hamster S-adenosylmethionine decarboxylase cDNA.

The nucleotide sequence of a cDNA encoding the proenzyme of hamster S-adenosylmethionine decarboxylase including 169 nucleotides of the 5' untranslated region has been determined. The deduced amino acid sequence shows a remarkable similarity to the human proenzyme with only seven differences out of 334 amino acids. The nucleotide sequence of the 5' untranslated region showed 93% homology with the corresponding rat and human sequences suggesting that this region may play an important role in the regulation of S-adenosylmethionine decarboxylase expression.     

Quantitation of S-adenosylmethionine decarboxylase protein

A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32 000, (b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigen had a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in the amount of enzyme protein measured both by titration of the active site and by radioimmunoassay.     

Structural biology of S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and a subject of many structural and biochemical investigations for anti-cancer and anti-parasitic therapy. The enzyme undergoes an internal serinolysis reaction as a post-translational modification to generate the active site pyruvoyl group for the decarboxylation process. The crystal structures of AdoMetDC from Homo sapiens, Solanum tuberosum, Thermotoga maritima, and Aquifex aeolicus have been determined. Numerous crystal structures of human AdoMetDC and mutants have provided insights into the mechanism of autoprocessing, putrescine activation, substrate specificity, and inhibitor design to the enzyme. The comparison of the human and potato enzyme with the T. maritima and A. aeolicus enzymes supports the hypothesis that the eukaryotic enzymes evolved by gene duplication and fusion. The residues implicated in processing and activity are structurally conserved in all forms of the enzyme, suggesting a divergent evolution of AdoMetDC.     

S-adenosylmethionine decarboxylase of corn seedlings

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been purified 500-fold in 30% yield from the extract of etiolated corn seedlings (cv. Golden Crossbantam Bell). This preparation had a molecular weight of approximately 25,000. The K_m value was 5 micromolar for S-adenosylmethionine. Methylglyoxal bis(guanylhydrazone), hydroxylamine, and sulfhydryl reagents (such as p-hydroxymercuriphenylsulfonate and N-ethylmaleimide) were effective inhibitors of this enzyme. Germination of corn seed was accompanied by a rapid increase in enzyme activity and maximum activity occurred in 5-day-old seedlings.     

Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.

S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.     

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Subcellular distribution of S-adenosylmethionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmethionine separate from synthesis of spermidine.

The activity of S-adenosylmethionine decarboxylase in rat liver homogenates is localized chiefly in the crude nuclear fraction, probably associated with membrane fragments, with the remainder in the supernatant fraction. This distribution is not paralleled by the activity of the cytoplasmic enzyme, lactate dehydrogenase. The spermidine-synthesizing activity of whole homogenate is recovered entirely in the supermidine-synthesizing activity of whole homogenate is recovered entirely in the supernatant fraction. Measurement of various kinetic parameters in crude fractions provided not positive evidence for isozymes of S-adenosylmethionine decarboxylase. Some species do not possess a sedimentable fraction of S-adenosylmethionine decarboxylase activity in liver. In those species all activity present in the whole homogenate of liver is released into the supernatant fraction.     

An effective inhibitor of S-adenosylmethionine decarboxylase

New analogues of cAMP, namely 2'-phosphate of cAMP and its esters, were obtained by direct phosphorylation of N6-benzoyladenosine 3',5'-cyclic phosphate with 2-cyanoethyl phosphate in the presence of DCC followed by the removal of protecting groups.     

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.