Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

CEA CNRS Université Paris Saclay Institute for Integrative Biology of the Cell F 91198 Gif sur Yvette France

Czech Advanced Technology and Research Institute Palacký University Šlechtitelů 27 CZ 78371 Olomouc Czech Republic

Department of Experimental Biology Faculty of Science Palacký University Šlechtitelů 27 Olomouc CZ 78371 Czech Republic

Department of Genetic Resources for Vegetables Medicinal and Special Plants Centre of the Region Haná for Biotechnological and Agricultural Research Crop Research Institute Šlechtitelů 29 CZ 78371 Olomouc Czech Republic

INRAE AgroParisTech Université Paris Saclay Institut Jean Pierre Bourgin Route de Saint Cyr F 78026 Versailles France

Isotope Laboratory Institute of Experimental Botany The Czech Academy of Sciences Vídeňská 1083 CZ 14220 Prague Czech Republic

Zobrazit více v PubMed

Abdelhakim, L.O.A., Mendanha, T., Palma, C.F.F., Vrobel, O., Štefelová, N., Ćavar Zeljković, S. et al. (2022) Elevated CO2 improves the physiology but not the final yield in spring wheat genotypes subjected to heat and drought stress during anthesis. Frontiers in Plant Science, 13, 824476.

Adio, A.M., Casteel, C.L., De Vos, M., Kim, J.H., Joshi, V., Li, B. et al. (2011) Biosynthesis and defensive function of Nδ-acetylornithine, a jasmonate-induced Arabidopsis metabolite. Plant Cell, 23, 3303-3318.

Ahou, A., Martignago, D., Alabdallah, O., Tavazza, R., Stano, P., Macone, A. et al. (2014) A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines. Journal of Experimental Botany, 65, 1585-1603.

Batista-Silva, W., Heinemann, B., Rugen, N., Nunes-Nesi, A., Araújo, W.L., Braun, H.P. et al. (2019) The role of amino acid metabolism during abiotic stress release. Plant, Cell & Environment, 42, 1630-1644.

Bewley, M.C., Graziano, V., Jiang, J., Matz, E., Studier, F.W., Pegg, A.E. et al. (2006) Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target. Proceedings of the National Academy of Sciences of the United States of America, 103, 2063-2068.

Bolkenius, F.N. & Seiler, N. (1981) Acetylderivatives as intermediates in polyamine catabolism. The International Journal of Biochemistry, 13, 287-292.

Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W. et al. (2011) BUSTER version 2.1.0. Cambridge, UK: Global Phasing Ltd.

Bulaj, G., Kortemme, T. & Goldenberg, D.P. (1998) Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry, 37, 8965-8972.

Castresana, J. (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540-552.

Cavallini, D., Ricci, G., Duprè, S., Pecci, L., Costa, M., Matarese, R.M. et al. (1991) Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role. European Journal of Biochemistry, 202, 217-223.

Ćavar Zeljković, S., Aucique-Perez, C.E., Stefelova, N. & De Diego, N. (2022) Optimizing growing conditions for hydroponic farming of selected medicinal and aromatic plants. Food Chemistry, 375, 131845.

Coleman, C.S., Huang, H. & Pegg, A.E. (1996) Structure and critical residues at the active site of spermidine/spermine N1-acetyltransferase. Biochemical Journal, 316, 697-701. https://doi.org/10.1042/bj3160697

Coleman, C.S., Stanley, B.A., Jones, A.D. & Pegg, A.E. (2004) Spermidine/spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine and is not involved in polyamine metabolism. The Biochemical Journal, 384, 139-148.

Della Ragione, F. & Pegg, A.E. (1983) Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase. The Biochemical Journal, 213, 701-706.

Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792-1797.

Emsley, P. & Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica. Section D, Biological Crystallography, 60, 2126-2132.

Federico, R., Ercolini, L., Laurenzi, M. & Angelini, R. (1996) Oxidation of acetylpolyamines by maize polyamine oxidase. Phytochemistry, 43, 339-341.

Forouhar, F., Lee, I.S., Vujcic, J., Vujcic, S., Shen, J., Vorobiev, S.M. et al. (2005) Structural and functional evidence for Bacillus subtilis PaiA as a novel N1-spermidine/spermine acetyltransferase. The Journal of Biological Chemistry, 280, 40328-40336.

Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology, 59, 307-321.

Hamana, K. & Matsuzaki, S. (1985) Distinct difference in the polyamine compositions of bryophyta and pteridophyta. Journal of Biochemistry, 97, 1595-1601.

Han, B.W., Bingman, C.A., Wesenberg, G.E. & Phillips, G.N., Jr. (2006) Crystal structure of Homo sapiens thialysine Nε-acetyltransferase (HsSSAT2) in complex with acetyl coenzyme A. Proteins, 64, 288-293.

Hegde, S.S., Chandler, J., Vetting, M.W., Yu, M. & Blanchard, J.S. (2007) Mechanistic and structural analysis of human spermidine/spermine N1-acetyltransferase. Biochemistry, 46, 7187-7195.

Hennion, F., Bouchereau, A., Gauthier, C., Hermant, M., Vernon, P. & Prinzing, A. (2012) Variation in amine composition in plant species: how it integrates macroevolutionary and environmental signals. American Journal of Botany, 99, 36-45.

Hickman, A.B., Namboodiri, M.A., Klein, D.C. & Dyda, F. (1999) The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 a resolution with a bisubstrate analog. Cell, 97, 361-369.

Higashi, K., Ishigure, H., Demizu, R., Uemura, T., Nishino, K., Yamaguchi, A. et al. (2008) Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli. Journal of Bacteriology, 190, 872-878.

Ho, B.K. & Gruswitz, F. (2008) HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Structural Biology, 8, 49.

Jammes, F., Leonhardt, N., Tran, D., Bousserouel, H., Véry, A.A., Renou, J.P. et al. (2014) Acetylated 1,3-diaminopropane antagonizes abscisic acid-mediated stomatal closing in Arabidopsis. The Plant Journal, 79, 322-333.

Jänne, J., Alhonen, L., Pietilä, M., Keinänen, T.A., Uimari, A., Hyvönen, M.T. et al. (2006) Genetic manipulation of polyamine catabolism in rodents. Journal of Biochemistry, 139, 155-160.

Jell, J., Merali, S., Hensen, M.L., Mazurchuk, R., Spernyak, J.A., Diegelman, P. et al. (2007) Genetically altered expression of spermidine/spermine N1-acetyltransferase affects fat metabolism in mice via acetyl-CoA. The Journal of Biological Chemistry, 282, 8404-8413.

Joghee, N.N. & Jayaraman, G. (2014) Metabolomic characterization of halophilic bacterial isolates reveals strains synthesizing rare diaminoacids under salt stress. Biochimie, 102, 102-111.

Jun, D.Y., Rue, S.W., Han, K.H., Taub, D., Lee, Y.S., Bae, Y.S. et al. (2003) Mechanism underlying cytotoxicity of thialysine, lysine analog, toward human acute leukemia Jurkat T cells. Biochemical Pharmacology, 66, 2291-2300.

Kabsch, W. (2010) XDS. Acta Crystallographica. Section D, Biological Crystallography, 66, 125-132.

Kakegawa, T., Guo, Y., Chiba, Y., Miyazaki, T., Nakamura, M., Hirose, S. et al. (1991) Effect of acetylpolyamines on in vitro protein synthesis and on the growth of a polyamine-requiring mutant of Escherichia coli. Journal of Biochemistry, 109, 627-631.

Kim, D.W., Watanabe, K., Murayama, C., Izawa, S., Niitsu, M., Michael, A.J. et al. (2014) Polyamine Oxidase5 regulates Arabidopsis growth through thermospermine oxidase activity. Plant Physiology, 165, 1575-1590.

Kiyota, E., Pena, I.A. & Arruda, P. (2015) The saccharopine pathway in seed development and stress response of maize. Plant, Cell & Environment, 38, 2450-2461.

Kopečný, D., Končitíková, R., Tylichová, M., Vigouroux, A., Moskalíková, H., Soural, M. et al. (2013) Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate. The Journal of Biological Chemistry, 288, 9491-9507.

Korasick, D.A., Končitíková, R., Kopečná, M., Hájková, E., Vigouroux, A., Moréra, S. et al. (2019) Structural and biochemical characterization of aldehyde dehydrogenase 12, the last enzyme of proline catabolism in plants. Journal of Molecular Biology, 431, 576-592.

Landry, J. & Sternglanz, R. (2003) Yeast Fms1 is a FAD-utilizing polyamine oxidase. Biochemical and Biophysical Research Communications, 303, 771-776.

Lou, Y.R., Bor, M., Yan, J., Preuss, A.S. & Jander, G. (2016) Arabidopsis NATA1 acetylates putrescine and decreases defense-related hydrogen peroxide accumulation. Plant Physiology, 171, 1443-1455.

Lu, L., Berkey, K.A. & Casero, R.A., Jr. (1996) RGFGIGS is an amino acid sequence required for acetyl coenzyme A binding and activity of human spermidine/spermine N1acetyltransferase. The Journal of Biological Chemistry, 271, 18920-18924.

Mamont, P.S., Seiler, N., Siat, M., Joder-Ohlenbusch, A.M. & Knödgen, B. (1981) Metabolism of acetyl derivatives of polyamines in cultured polyamine-deficient rat hepatoma cells. Medical Biology, 59, 347-353.

Mattioli, R., Pascarella, G., D'Incà, R., Cona, A., Angelini, R., Morea, V. et al. (2022) Arabidopsis N-acetyltransferase activity 2 preferentially acetylates 1,3-diaminopropane and thialysine. Plant Physiology and Biochemistry, 170, 123-132.

McCloskey, D.E. & Pegg, A.E. (2003) Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1,N11-bis(ethyl)norspermine. The Journal of Biological Chemistry, 278, 13881-13887.

Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S. & Steinegger, M. (2022) ColabFold: making protein folding accessible to all. Nature Methods, 19, 679-682.

Montemayor, E.J. & Hoffman, D.W. (2008) The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis. Biochemistry, 47, 9145-9153.

Müller, B., Fastner, A., Karmann, J., Mansch, V., Hoffmann, T., Schwab, W. et al. (2015) Amino acid export in developing Arabidopsis seeds depends on UmamiT facilitators. Current Biology, 25, 3126-3131.

Neuwald, A.F. & Landsnan, D. (1997) GCN5-related histone N-acetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein. Proceedings of the National Academy of Sciences of the United States of America, 51, 154-155.

Pegg, A.E. (2008) Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator. American Journal of Physiology. Endocrinology and Metabolism, 294, E995-E1010.

Perez-Leal, O. & Merali, S. (2012) Regulation of polyamine metabolism by translational control. Amino Acids, 42, 611-617.

Pietilä, M., Alhonen, L., Halmekytö, M., Kanter, P., Jänne, J. & Porter, C.W. (1997) Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. The Journal of Biological Chemistry, 272, 18746-18751.

Scheibner, K.A., De Angelis, J., Burley, S.K. & Cole, P.A. (2002) Investigation of the roles of catalytic residues in serotonin N-acetyltransferase. The Journal of Biological Chemistry, 277, 18118-18126.

Storoni, L.C., McCoy, A.J. & Read, R.J. (2004) Likelihood-enhanced fast rotation functions. Acta Crystallographica. Section D, Biological Crystallography, 60, 432-438.

Tabor, C.W., Tabor, H. & Bachrach, U. (1964) Identification of the aminoaldehydes produced by the oxidation of spermine and spermidine with purified plasma amine oxidase. The Journal of Biological Chemistry, 239, 2194-2203.

Tavladoraki, P., Cona, A., Federico, R., Tempera, G., Viceconte, N., Saccoccio, S. et al. (2012) Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants. Amino Acids, 42, 411-426.

Tickle, I.J., Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C. et al. (2018) STARANISO. Cambridge, UK: Global Phasing Ltd. http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi

Toumi, I., Pagoulatou, M.G., Margaritopoulou, T., Milioni, D. & Roubelakis-Angelakis, K.A. (2019) Genetically modified heat shock Protein90s and polyamine oxidases in Arabidopsis reveal their interaction under heat stress affecting polyamine acetylation, oxidation and homeostasis of reactive oxygen species. Plants, 8, 323.

Uemura, T., Yerushalmi, H.F., Tsaprailis, G., Stringer, D.E., Pastorian, K.E., Hawel, L., 3rd et al. (2008) Identification and characterization of a diamine exporter in colon epithelial cells. The Journal of Biological Chemistry, 283, 26428-26435.

Vujcic, S., Halmekyto, M., Diegelman, P., Gan, G., Kramer, D.L., Janne, J. et al. (2000) Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. The Journal of Biological Chemistry, 275, 38319-38328.

Wallace, H.M., Fraser, A.V. & Hughes, A. (2003) A perspective of polyamine metabolism. The Biochemical Journal, 376, 1-14.

Winter, G., Todd, C.D., Trovato, M., Forlani, G. & Funck, D. (2015) Physiological implications of arginine metabolism in plants. Frontiers in Plant Science, 6, 534.

Yamaguchi, K., Takahashi, Y., Berberich, T., Imai, A., Miyazaki, A., Takahashi, T. et al. (2006) The polyamine spermine protects against high salt stress in Arabidopsis thaliana. FEBS Letters, 580, 6783-6788.

Zhao, F., Song, C.P., He, J. & Zhu, H. (2007) Polyamines improve K+/Na+ homeostasis in barley seedlings by regulating root ion channel activities. Plant Physiology, 145, 1061-1072.

Zhu, Y.Q., Zhu, D.Y., Yin, L., Zhang, Y., Vonrhein, C. & Wang, D.C. (2006) Crystal structure of human spermidine/spermine N1-acetyltransferase (hSSAT): the first structure of a new sequence family of transferase homologous superfamily. Proteins, 63, 1127-1131.

Comprehensive LC-MS/MS analysis of nitrogen-related plant metabolites

The loss-of-function of AtNATA2 enhances AtADC2-dependent putrescine biosynthesis and priming, improving growth and salinity tolerance in Arabidopsis