Identification of the amino acid residues conferring substrate specificity upon Selenomonas ruminantium lysine decarboxylase

Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar K(m) and Vmax. Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).     

Characterization of a counterpart to Mammalian ornithine decarboxylase antizyme in prokaryotes

The degradation of mammalian ornithine decarboxylase (ODC) (EC 4.1.1.17) by 26 S proteasome, is accelerated by the ODC antizyme (AZ), a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryotes, AZ has not been found. Previously, we found that in Selenomonas ruminantium, a strictly anaerobic and Gram-negative bacterium, a drastic degradation of lysine decarboxylase (LDC; EC 4.1.1.18), which has decarboxylase activities toward both L-lysine and L-ornithine with similar K(m) values, occurs upon entry into the stationary phase of cell growth by protease together with a protein of 22 kDa (P22). Here, we show that P22 is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium LDC by a 26 S proteasome. (iii) S. ruminantium LDC degradation is also enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ. In this report, we also show that P22 is a ribosomal protein of S. ruminantium.     

Novel Characteristics of Selenomonas ruminantium Lysine Decarboxylase Capable of Decarboxylating Both L-Lysine and L-Ornithine

Lysine decarboxylase (LDC; EC 4.1.1.18) of Selenomonas ruminantium is a constitutive enzyme and is involved in the synthesis of cadaverine, which is an essential constituent of the peptidoglycan for normal cell growth. We purified the S. ruminantium LDC by an improved method including hydrophobic chromatography and studied the fine characteristics of the enzyme. Kinetic study of LDC showed that S. ruminanitum LDC decarboxylated both L-lysine and L-ornithine with similar K _m and the decarboxylase activities towards both substrates were competitively and irreversibly inhibited by DL-α-difluoromethylornithine, which is a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17). We also showed a drastic descent of LDC activity owing to the degradation of LDC at entry into the stationary phase of cell growth.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Gene cloning and molecular characterization of lysine decarboxylase from Selenomonas ruminantium delineate its evolutionary relationship to ornithine decarboxylases from eukaryotes

Lysine decarboxylase (LDC; EC 4.1.1.18) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both L-lysine and L-ornithine with similar K(m) and V(max) values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063-1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17), including the mouse, Saccharomyces cerevisiae, Neurospora crassa, Trypanosoma brucei, and Caenorhabditis elegans enzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved in S. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward both L-lysine and L-ornithine with a substrate specificity ratio of 0.83 (defined as the k(cat)/K(m) ratio obtained with L-ornithine relative to that obtained with L-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.     

Identification of a 22-kDa protein required for the degradation of Selenomonas ruminantium lysine decarboxylase by ATP-dependent protease

In Selenomonas ruminantium, a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, a rapid degradation of lysine decarboxylase (LDC) occurred on entry into the stationary phase of cell growth. Here, we identified a 22-kDa protein as a stimulating factor for the degradation of LDC, which was catalyzed by ATP-dependent protease(s) in S. ruminantium. The purified 22-kDa protein preparation itself had no degradation activity towards LDC but it was required for the degradation of LDC by ATP-dependent proteases in a cell-free system. The 22-kDa protein had similar biochemical and biophysical characteristics to those of antizyme, the regulator for the degradation of mammalian ODC, which had been reported only in mammalian cells. From the sequencing data of the N-terminal 30 amino acid residues of the 22-kDa protein preparation, 22-kDa protein was found to be a new protein which was distinguished from antizyme. This is the first report of the presence of an antizyme-like regulator protein in a prokaryote.     

Molecular dissection of the Selenomonas ruminantium cell envelope and lysine decarboxylase involved in the biosynthesis of a polyamine covalently linked to the cell wall peptidoglycan layer

The wild type of Selenomonas ruminantium subsp. lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells are grown in a lactate medium. This bacterium is characterized by a unique structure of the cell envelope and a novel lysine decarboxylase and its regulatory protein. In the first part of this article, we will refer to the chemical structure of phospholipid and lipopolysaccharide in the cell membranes of this bacterium compared with that from the general Gram-negative bacteria for understanding their biological functions. S. ruminantium has neither free nor bound forms of Braun lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. However, S. ruminantium has cadaverine, which links covalently to the peptidoglycan as a pivotal constituent for the cell division. In the second part of this article, we will refer to the chemical structure of the cadaverine-containing peptidoglycan, its biosynthesis, and the biological function. In the third part of this article, we will depict the molecular cloning of the genes encoding S. ruminanitum lysine decarboxylase (LDC) and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, and the molecular dissection of these proteins for understanding the regulation of cadaverine biosynthesis. Finally, we will illustrate a proposed structure of the cell envelope, a processes of biosynthesis of the cadaverine-containing peptidoglycan layer, and the LDC degradation mechanism in S. ruminantium, on the basis of the analyses of the cell envelope components, the results from the in vitro experiments on the biosynthesis of the peptidoglycan layer, and the current status of the knowledge on LDC and P22 in this organism.     

Cadaverine covalently linked to a peptidoglycan is an essential constituent of the peptidoglycan necessary for the normal growth in Selenomonas ruminantium

Cadaverine links covalently to the D-glutamic acid residue of the peptidoglycan in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium (Kamio, Y., Itoh, Y., and Terawaki, Y. (1981) J. Bacteriol. 146, 49-53). This report clarifies a physiological function of cadaverine in this organism by using DL-alpha-difluoromethyllysine, which had previously been shown to be a selective irreversible inhibitor of lysine decarboxylase of Mycoplasma dispar (P?s?, H., MaCann, P.P., Tanskanen, R., Bey, P., and Sjoerdsma, A. (1984) Biochem. Biophys. Res. Commun. 125, 205-210). DL-alpha-Difluoromethyllysine is now shown to be a potent and irreversible inhibitor of lysine decarboxylase of S. ruminantium in vitro; however, it did not inhibit the transfer of cadaverine to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan. DL-alpha-Difluoromethyllysine at 5 mM markedly inhibited the growth of the bacterium and caused rapid cell lysis. Immediately before the cell lysis, almost all cells became swollen, and such cells showed a loosened envelope structure when studied by electron microscopy. The peptidoglycan prepared from the DL-alpha-difluoromethyllysine-treated cells did not have covalently linked cadaverine. The growth inhibition by DL-alpha-difluoromethyllysine was completely reversed by adding cadaverine (1 mM) to the medium. Furthermore, the exogenous cadaverine was exclusively incorporated into the peptidoglycan in the presence of DL-alpha-difluoromethyllysine (5 mM), and a normal peptidoglycan was synthesized. The cell lysis and the formation of an abnormal cell structure were completely prevented by cadaverine added to the medium. We conclude that cadaverine covalently linked to the peptidoglycan in S. ruminantium is an essential constituent of the peptidoglycan and is required for cell surface integrity and the normal growth of S. ruminantium.     

Participation of the enzymes involved in the biosynthesis of biogenic amines in biochemical interactions between wheat (Triticum aestivum; Poaceae) and bird cherry-oat aphid (Rhopalosiphum padi; Aphididae)

The studies concerned changes in the activities of ornithine decarboxylase (ODC), lysine decarboxylase (LDC) and tyrosine decarboxylase (TyDC) in tissues of wheat (Triticum aestivum L.) infested with bird cherry-oat aphid (Rhopalosiphum padi L.).Obtained results showed that the activities of the enzymes were stimulated in the less susceptible wheat Kontesa cv. infested by the aphids. In the case of the more susceptible Tonacja cv., on most occasions a decrease in the enzyme activities occurred. Such responses were especially clear for TyDC in both analysed cvs., and for LDC and ODC in the case of Kontesa cv. Thus it may be concluded that amino acid decarboxylation plays an important part in the biochemical defence developed in wheat tissues in response to R. padi infestation. The changes in the activities of the decarboxylases were dependent on the wheat genotype as well as the duration of the infestation.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Changes in activity of lysine decarboxylase in winter triticale in response to grain aphid feeding

Changes in lysine decarboxylase (LDC) activity caused by Sitobion avenae (F.) feeding on two winter triticale cultivars (cvs) were studied. The aphid fecundity and values of intrinsic rate of natural increase showed that cv Witon was less susceptible to S. avenae than cv Tornado. The grain aphid feeding on more susceptible triticale caused a decrease in the LDC activity, with exceptions of root tissues after two weeks of the feeding. In case of less susceptible cv Witon reduction of the LDC activity was observed only during initial period of S. avenae feeding. Later the aphid infestation induced activity of the LDC within tissues of cv Witon.     

Induction of de-novo synthesis of tryptophan decarboxylase in cell suspensions of Catharanthus roseus

Tryptophan decarboxylase (EC 4.2.1.27) is synthesized de-novo by Catharanthus roseus cells shortly after the cells have been transferred into culture medium in which monoterpenoid indole alkaloids are formed. The enzyme production, monitored by in-vivo labelling with [³⁵S]methionine and immunoprecipitation, precedes the apparent maximal enzyme activity by 10–12 h. From the time course of the descending enzyme activity after induction, a half-life of 21 h for tryptophan decarboxylase in C. roseus cell suspensions is calculated. A comparison of the polyadenylated-RNA preparations from C. roseus cells indicates that mRNA activity for tryptophan decarboxylase is only detected in cells grown in the production medium. The importance of tryptophan decarboxylase induction with respect to the accumulation of th corresponding alkaloids is discussed.Abbreviation TDC tryptophan decarboxylase     

Changes in amino acid decarboxylation in maize (Zea mays; Poaceae) tissues in response to bird cherry-oat aphid (Rhopalosiphum padi; Aphididae) infestation

The study was focused on changes in the activities of ornithine decarboxylase (ODC), lysine decarboxylase (LDC) and tyrosine decarboxylase (TyDC) in maize seedlings infested by bird cherry-oat aphid (Rhopalosiphum padi L.).Obtained results showed that the aphid infestation induced LDC activity strongly within tissues of less aphid-settled maize (Waza cv.) during the first week of the infestation in comparison to the control plants and the activity was suppressed after two weeks. However, TyDC activity fluctuated under the aphid infestation. In relation to the control, the enzyme activity was inhibited on the first day, activated after the first week and reduced again two weeks into the experiment. A significant reduction in ODC activity was also observed in seedlings of aphid-infested maize Waza cv. during the first week. In a more susceptible maize cv. (Złota Karłowa), an increase in LDC and TyDC activities in relation to the control and a simultaneous decrease in ODC activity were noted after the first day of the infestation. After one week of aphid attack, TyDC activity was induced and ODC was inhibited, whereas after two weeks ODC activity was decreased with a simultaneous increase in LDC activity in the Złota Karłowa seedlings.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

Ornithine decarboxylase in difluoromethylornithine-resistant mouse lymphoma cells. Two-dimensional gel analysis of synthesis and turnover

Variant S49 mouse lymphoma cells with increased ornithine decarboxylase activity were obtained by selecting for resistance to alpha-difluoromethylornithine (DFMO), a specific inhibitor of the enzyme. Ornithine decarboxylase was identified as a specifically immunoprecipitable polypeptide that was made at an increased rate in the variant cells. Ornithine decarboxylase was also identified on a two-dimensional gel as a metabolically labeled polypeptide of Mr approximately 55,000 which was synthesized at an increased rate in two independently selected variants. Synthesis of this polypeptide was further augmented by treatment of cells with inhibitors of ornithine decarboxylase activity. The charge of the polypeptide was altered by treatment of either cells or cellular extracts with DFMO, a suicide substrate which binds covalently to the enzyme. This charge alteration and the inactivation of ornithine decarboxylase showed the same dependence on DFMO concentration and both effects were prevented by addition of either ornithine or putrescine. Pulse-chase experiments showed that the half-life of the ornithine decarboxylase polypeptide in these variant cells was 45 min. We conclude that ornithine decarboxylase is made at an increased rate in the resistant variants and that the polypeptide turns over rapidly.     

Polyamine biosynthesis and accumulation during the G1 to S phase transition

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G₁ phase of the cell cycle and decreases as the cells progress into S phase. S-adenosyl-L-methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G₁/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G₁ and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid-G₁ phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.