Polyamines Regulate Strawberry Fruit Ripening by Abscisic Acid, Auxin, and Ethylene.

Polyamines (PAs) participate in many plant growth and developmental processes, including fruit ripening. However, it is not clear whether PAs play a role in the ripening of strawberry (Fragaria ananassa), a model nonclimacteric plant. Here, we found that the content of the PA spermine (Spm) increased more sharply after the onset of fruit coloration than did that of the PAs putrescine (Put) or spermidine (Spd). Spm dominance in ripe fruit resulted from abundant transcripts of a strawberry S-adenosyl-l-Met decarboxylase gene (FaSAMDC), which encodes an enzyme that generates a residue needed for PA biosynthesis. Exogenous Spm and Spd promoted fruit coloration, while exogenous Put and a SAMDC inhibitor inhibited coloration. Based on transcriptome data, up- and down-regulation of FaSAMDC expression promoted and inhibited ripening, respectively, which coincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness, anthocyanin content, sugar content, polyamine content, auxin (indole-3-acetic acid [IAA]) content, abscisic acid (ABA) content, and ethylene emission. Using isothermal titration calorimetry, we found that FaSAMDC also had a high enzymatic activity with a Kd of 1.7 × 10-3 m In conclusion, PAs, especially Spm, regulate strawberry fruit ripening in an ABA-dominated, IAA-participating, and ethylene-coordinated manner, and FaSAMDC plays an important role in ripening.

[Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale].

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe-4S](2+) cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.

Identification and assay of allosteric regulators of S-adenosylmethionine decarboxylase.

Polyamine biosynthesis is extensively regulated in cells by multiple mechanisms, including regulation of enzyme activity posttranslationally. The identified regulatory factors include both small molecules and regulatory proteins, and the mechanisms vary in different species across the evolutionary tree. Based on this diversity of mechanism, it is likely that regulatory factors of the pathway remain unidentified in many species. This article focuses on methods for identifying novel regulatory factors of polyamine biosynthesis as illustrated by the discovery of a novel protein activator of the key biosynthetic enzyme S-adenosylmethionine decarboxylase in the protozoan trypanosomatid parasites.

Cloning, expression and purification of human S-adenosylmethionine decarboxylase gene alpha subunit.

S-adenosylmethionine decarboxylase (SAMDC) is an essential enzyme for the synthesis of spermidine and spermine in the biosynthetic pathway of polyamines. The total RNA was extracted from colon cancer tissue and amplified by reverse-transcription PCR with two primers, which span the coding region of SAMDC alpha subunit. Clone vector pMD18-T-SAMDC-alpha was successfully constructed by using T-A clone technique. pMD18-T-SAMDC-alpha and pTriEx-4 were digested by NcoI and XhoI double enzymes. The purified SAMDC-alpha fragment was subcloned into the expression vector pTriEx-4 to construct the prokaryotic expression plasmid pTriEx-4-SAMDC-alpha. The recombinant plasmid pTriEx-4-SAMDC-alpha was transformed into competence E. coli JM109 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 32 kDa exogenous protein was observed on the SDS-PAGE. The protein was verified by Western blot with anti His.Tag monoclonal antibody. The fusion protein including 6 x His.Tag was purified by Ni-NTA chromatographic column. Then, the purified protein can be applied for further research of the immunity of SAMDC.

In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes.

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed in E. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the M. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.

In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase.

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

Mechanistic studies of the processing of human S-adenosylmethionine decarboxylase proenzyme. Isolation of an ester intermediate.

Human S-adenosylmethionine decarboxylase is synthesized as a proenzyme that undergoes an autocatalytic cleavage reaction generating the alpha and beta subunits and forming the pyruvate prosthetic group, which is derived from an internal Ser residue (Ser-68). The mechanism of this processing reaction was studied using site-directed mutagenesis of conserved residues (His-243 and Ser-229) located close to the cleavage site. Mutant S229A failed to process, and mutant S229C cleaved very slowly, whereas mutant S229T processed normally, suggesting that the hydroxyl group of residue 229 is required for the processing reaction where Ser-229 may act as a proton acceptor. Mutant His-243A cleaved very slowly, forming a small amount of the correctly processed pyruvoyl enzyme but a much larger proportion of the alpha subunit with an amino-terminal Ser. The cleavage to form the latter was greatly enhanced by hydroxylamine. This result suggests that the N-O acyl shift needed for ester formation occurs normally in this mutant but that the next step, which is a beta-elimination reaction leading to the two subunits, does not occur. His-243 may therefore act as the basic residue that extracts the hydrogen of the alpha-carbon of Ser-68 in the ester in order to facilitate this reaction. The availability of the recombinant H243A S-adenosylmethionine decarboxylase proenzyme provides a useful model system to examine the processing reaction in vitro and test the design of specific inactivators aimed at blocking the production of the pyruvoyl prosthetic group.

Cloning and kinetic characterization of the Trypanosoma cruzi S-adenosylmethionine decarboxylase.

The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (alpha) and 10 kDa (beta). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s(-1). The model derived Km for AdoMet is 0.05 mM in the absence of putrescine and the model-derived Kd for putrescine binding to free enzyme is 2.5 mM. The Km for AdoMet increases by alpha 2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine.

A new S-adenosylmethionine decarboxylase from soybean axes.

A new active S-adenosylmethionine decarboxylase (EC 4.1.1.50) (SAMDC II) was extracted from soybean (Glycine max) axes. The enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone) (MGBG)-Sepharose 6B chromatographies. The molecular weight of the native enzyme was 110,000, while the subunit molecular weights were 66,000 and 58,000, indicating a heterodimeric structure. The Km value of the enzyme for S-adenosylmethionine was 16 microM, which is two times higher than that of previously reported S-adenosylmethionine decarboxylase (SAMDC I) (8.1 microM). The specific activity of SAMDC II during the seed growth increased rapidly and reached its maximum on the second day after germination whereas that of SAMDC I reached its peak on the fourth day. MGBG was shown to inhibit SAMDC II competitively like SAMDC I. Carbonyl and sulfhydryl group specific reagents modified SAMDC II, resulting in the loss of enzymatic activity. Agmatine, the product of arginine decarboxylation catalyzed by arginine decarboxylase, inhibited the SAMDC II competitively (Ki = 40 microM) while it inhibited the SAMDC II non-competitively (Ki = 600 mM). The possible role of the chronological appearance of SAMDC II and SAMDC I, and properties of the enzyme are briefly discussed in connection with polyamine biosynthesis in soybean axes.

Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.

Characterization of S-adenosylmethionine decarboxylase induced by human cytomegalovirus infection.

Infection of human diploid embryonic lung (MRC5) cells by human cytomegalovirus (HCMV), strain AD169, increased the activity of a key enzyme in the synthesis of polyamines: S-adenosylmethionine decarboxylase (E.C. 4.1.1.50). The initial peak of S-adenosylmethionine decarboxylase activity occurred about 15 h postinfection. S-Adenosylmethionine decarboxylase was purified using a highly specific affinity chromatography step from HCMV-infected and control uninfected MRC5 cells. No difference was found between the two enzymes in their stability to heat or effect of pH on activity. Both enzymes were activated only by putrescine. The appKm for S-adenosylmethionine for the virus-induced enzyme was 1.7 times higher than the appKm for the control enzyme. The most dramatic difference observed was in the effect of high salt concentration on enzyme activity. S-Adenosylmethionine decarboxylase from HCMV-infected cells was unaffected by 0.8 M NaCl, whereas the enzyme from uninfected cells was inhibited by 50% at 0.45 M NaCl and was significantly inhibited at a concentration of 0.8 M NaCl. Thus, different forms of S-adenosylmethionine decarboxylase probably exist in infected and uninfected MRC5 cells.

S-adenosyl-L-methionine decarboxylase of Acanthamoeba castellanii (Neff): purification and properties.

S-Adenosyl-L-methionine decarboxylase (AdoMetDC) has been purified to near homogeneity from the Neff strain of Acanthamoeba castellanii. The holoenzyme molecular mass is 88.8 kDa, including two copies each of a 32.8 kDa alpha-subunit and a 10-15 kDa beta-subunit. The alpha-subunit contains the active site. It has an N-terminal pyruvoyl group, and the first 19 amino acids are 63 and 74% identical with comparable sequences from yeast and mammals, respectively. The apparent Km for S-adenosylmethionine (AdoMet) in the presence of 2 mM putrescine was 30.0 microM. The enzyme was stimulated 2-fold by putrescine, but was unaffected by spermidine. It was inhibited by the following anti-metabolites, listed with their Ki values: Berenil (0.17 microM), pentamidine (19.4 microM), propamidine (334 microM), hydroxystilbamidine (357 microM), methylglyoxal bis(guanylhydrazone) (604 microM) and ethidium bromide (1.3 mM). Activity of the enzyme fell to undetectable levels during cell differentiation (encystment).

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.

Irreversible inhibition of S-adenosylmethionine decarboxylase of Trypanosoma brucei brucei by S-adenosylmethionine analogues.

S-Adenosylmethionine analogues designed as active-site directed inhibitors were tested in vitro for their effects on S-adenosylmethionine decarboxylase (AdoMetDC) of Trypanosoma brucei brucei. These analogues contained a tertiary nitrogen atom in place of the sulfonium and had a side chain of variable length ending in a reactive group (hydrazino-, aminooxy-, hydrazido- or a methylnitrosourea). The hydrazino- derivatives were the most potent inhibitors with IC50 values in the range of 40-100 nM. The most active compound (IC50 of 0.04 microM) was 5'-deoxy-5'-[(2-hydrazinoethyl)-methylamino]adenosine (MHZEA). Addition of MHZEA produced a time-dependent inactivation with an apparent Ki of 0.4 microM, and the enzyme half-life at a saturating concentration of MHZEA was 0.4 min. Increasing the length of the side chain or changing the methyl group attached to the nitrogen to an ethyl group reduced the potency. Replacement of the hydrazino moiety with an aminooxy group resulted in about a 30- to 35-fold decrease in inhibition potency. However, the relative order of activities of these aminooxy analogues was similar to that found in the hydrazino series with 5'-deoxy-5'-[(2-aminooxyethyl)methylamino]adenosine (MAOEA), which had an IC50 of 1.3 microM, being the most active. The hydrazido analogs were even less effective with 5'-deoxy-5'-[(3-hydrazino-3-oxopropyl)-methylamino]adenosine, the best inhibitor, having an IC50 value of 8.7 microM. The methylnitrosourea derivatives were inactive. The inactivation of trypanosomal AdoMetDC with MHZEA or MAOEA was irreversible and was greatly stimulated by putrescine, a known activator of the enzyme, indicating that the compounds bind to the active site and form a covalent bond with the enzyme. These inhibitors may have considerable potential as chemotherapeutic agents against trypanosomiasis and other protozoal infections and may also be useful in studying the role of AdoMetDC in the regulation of polyamine levels in these organisms.

Purification of human S-adenosylmethionine decarboxylase expressed in Escherichia coli and use of this protein to investigate the mechanism of inhibition by the irreversible inhibitors, 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine.

Human S-adenosylmethionine decarboxylase (AdoMetDC) was expressed in high yield in Escherichia coli using the pIN-III(lppP-5) expression vector and purified to apparent homogeneity using affinity chromatography on methylglyoxal bis(guanylhydrazone)-Sepharose. The inactivation of the purified enzyme by 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine (MHZPA) was accompanied by an increase in absorbance at 260 nm of the large subunit. This increase was equivalent to the addition of 1 molecule of MHZPA. After digestion with the protease Lys-C, a peptide that contained the bound MHZPA was isolated and found to have the amino acid composition consistent with that expected from the amino terminus of the large subunit. These results indicate that MHZPA inactivates AdoMetDC by forming a hydrazone derivative at the pyruvate prosthetic group. Inactivation of AdoMetDC by 5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo) led to the appearance of a new peptide peak in the Lys-C protease digest. This peptide had the sequence ASMFVSK. This agrees with the expected sequence from the amino terminus, which is pyruvoyl-SMFVSK, with the exception that the pyruvate has been converted to alanine. Direct gas-phase sequencing of the large subunit of the enzyme also indicated the presence of alanine at the amino terminus after inactivation with AbeAdo. These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group. Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC.

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.

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.

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase.

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., Jänne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.