Publications

• Dual functioning of plant arginases provides a third route for putrescine synthesis.

  Patel J., Ariyaratne M., Ahmed S., Ge L., Phuntumart V., Kalinoski A., Morris P.F.

  Two biosynthetic routes are known for putrescine, an essential plant metabolite. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine, while a second route for putrescine biosynthesis utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzym ... >> More

  Two biosynthetic routes are known for putrescine, an essential plant metabolite. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine, while a second route for putrescine biosynthesis utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzymes, agmatine iminohydrolase (AIH) and N-carbamoyl putrescine aminohydrolase (NLP1) to complete this pathway. Here we show that plants can use ADC and arginase/agmatinase (ARGAH) as a third route for putrescine synthesis. Transformation of Arabidopsis thaliana ADC2, and any of the arginases from A. thaliana (ARGAH1, or ARGHA2) or the soybean gene Glyma.03g028000 (GmARGAH) into a yeast strain deficient in ODC, fully complemented the mutant phenotype. In vitro assays using purified recombinant enzymes of AtADC1 and AtARGAH2 were used to show that these enzymes can function in concert to convert arginine to agmatine and putrescine. Transient expression analysis of the soybean genes (Glyma.06g007500, ADC; Glyma.03g028000 GmARGAH) and the A. thaliana ADC2 and ARGAH genes in leaves of Nicotiana benthamiana, showed that these proteins are localized to the chloroplast. Experimental support for this pathway also comes from the fact that expression of AtARGAH, but not AtAIH or AtNLP1, is co-regulated with AtADC2 in response to drought, oxidative stress, wounding, and methyl jasmonate treatments. Based on the high affinity of ARGAH2 for agmatine, its co-localization with ADC2, and typically low arginine levels in many plant tissues, we propose that these two enzymes can be major contributors to putrescine synthesis in many A. thaliana stress responses. << Less

  Plant Sci. 262:62-73(2017) [PubMed] [EuropePMC]

• Isolation and characterization of a mutant of Escherichia coli blocked in the synthesis of putrescine.

  Hirshfield I.N., Rosenfeld H.J., Leifer Z., Maas W.K.

  A mutant of Escherichia coli is described which is defective in the conversion of arginine to putrescine. The activity of the enzyme agmatine ureohydrolase is greatly reduced, whereas the activity of the other two enzymes of the pathway, the constitutive arginine decarboxylase and the inducible ar ... >> More

  A mutant of Escherichia coli is described which is defective in the conversion of arginine to putrescine. The activity of the enzyme agmatine ureohydrolase is greatly reduced, whereas the activity of the other two enzymes of the pathway, the constitutive arginine decarboxylase and the inducible arginine decarboxylase, are within the normal range. The growth behavior of the mutant reflects the enzymatic block. It grows well in the absence of arginine, but only poorly in the presence of arginine. Under the former conditions, putrescine can be formed from ornithine as well as arginine, whereas under the latter conditions, because of feedback control, it can be formed only from arginine. << Less

  J Bacteriol 101:725-730(1970) [PubMed] [EuropePMC]

• The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.

  Goda S., Sakuraba H., Kawarabayasi Y., Ohshima T.

  Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene wa ... >> More

  Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine-HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co(2+), Ca(2+) and Mn(2+) for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110-145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 degrees C for 10 min, and a half of the activity was retained by incubation at 90 degrees C for 10 min. From a typical Michaelis-Menten type kinetics, an apparent K(m) value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics. << Less

  Biochim. Biophys. Acta 1748:110-115(2005) [PubMed] [EuropePMC]

• A new subfamily of agmatinases present in methanogenic Archaea is Fe(II) dependent.

  Miller D., Xu H., White R.H.

  Here we report that the Methanocaldococcus jannaschii enzyme derived from the MJ0309 gene is an Fe(II) dependent agmatinase (SpeB). This is the first report of an iron-dependent agmatinase. We demonstrate that aerobically isolated recombinant enzyme contains two disulfide bonds and only a trace am ... >> More

  Here we report that the Methanocaldococcus jannaschii enzyme derived from the MJ0309 gene is an Fe(II) dependent agmatinase (SpeB). This is the first report of an iron-dependent agmatinase. We demonstrate that aerobically isolated recombinant enzyme contains two disulfide bonds and only a trace amount of any metal and requires the presence of both dithiothreitol (DTT) and 4 equiv of Fe(II) for maximum activity. The DTT activation could be indicative of the presence of a redox system, which would regulate the activity of this as well as other enzymes in the methanogens. Site-directed mutagenesis of the four conserved cysteines C71, C136, C151, and C229 to alanine or serine showed that only the C71 and C151 mutants showed a significant drop in activity indicating that the disulfide bond responsible for regulating activity was likely between C136 and C229. We propose that the C71 and C151 cysteine thiols, produced by the DTT-dependent reduction of their disulfide, are two additional metal binding ligands that alter the metal specificity of the M. jannaschii agmatinase from Mn(II) to Fe(II). << Less

  Biochemistry 51:3067-3078(2012) [PubMed] [EuropePMC]

• First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3.

  Kawakami R., Sakuraba H., Ohmori T., Ohshima T.

  A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichi ... >> More

  A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction was markedly improved by co-expression with chaperone molecules. The purified enzyme retained its full activity after incubation at 80°C for at least 2 h in buffer (pH 7-10), making this enzyme the most thermostable amino acid racemase so far known. Besides the nine amino acids containing hydrophobic and aromatic amino acids previously reported (Kawakami et al., Amino Acids, 47, 1579-1587, 2015), the enzyme exhibited substantial activity toward Thr (about 42% of relative activity toward Phe) and showed no activity toward Arg, His, Gln, and Asn. The substrate specificity of this enzyme thus differs markedly from those of other known amino acid racemases. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. The high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids. << Less

  J. Biosci. Bioeng. 124:23-27(2017) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.