Aliphatic 1-Amino Acid Decarboxylase from Ferns (Filicopsida) Thomas Hartmann. Klaus Bax. and Renate Scholz Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendels- sohnstr. 1, D-3300 Braunschweig. Bundesrepublik Deutschland Z. Naturforsch. 39c, 2 4 -3 0 (1984); received Septem ber 26, 1983 Ferns, Filicopsida, Polypodium vulgare. Aliphatic 1-Amino Acid Decarboxylase, Occurrence and Distribution A screening of 27 fern species (Filicopsida) out of 9 families revealed that 25 species were able to decarboxylate 1-leucine to 3-methylbutylamine (isoamylamine). The enzyme of Polvpodium vulgäre has partially been purified and characterisized. All attempts to solubilize it from acetone preparations failed; however, approx. 50% of total activity could be extracted from dry material in the presence of detergents at high concentration. The soluble enzyme was purified 132-fold. 1-Methionine was found the best substrate followed by norvaline, leucine, norleucine, isoleucine, homocysteine, valine. It has been confirmed that these substrates are decarboxylated by a single enzyme. The pH-optimum was at pH 5.0 (particulate preparation) and pH 4.5 (soluble enzyme). Decarboxylation is dependent on pyridoxal-5'-phosphate (PLP). A strictly substrate dependent coenzyme dissociation was observed which could largely be prevented by addition of 2 -oxo-acids, such as glyoxylate or pyruvate. Apodecarboxylase prepared by prolonged substrate incubation was found to be extremely labile at pH 4.5 but stable at pH 6.5. A comparison of the fern enzyme with bacterial valine decarboxylase (EC 4.1.1.14) and leucine decarboxylase of red algae revealed great similarities especially in substrate specificity. It is suggested to unify these activities as “aliphatic 1-amino acid decarboxylase”. Introduction decarboxylase from ferns which displays similar properties as the algal and bacterial enzymes Lower aliphatic monoamines occur widely dis­ mentioned above. tributed in the plant kingdom [1]. Two alternative enzymatic routes of amine synthesis have been established: aldehyde amination and amino acid Materials and Methods decarboxylation. The first route catalyzed by an alanine: aldehyde aminotransferase is the common Plant material biosynthetic pathway for a wide range of amines in Most fern species were taken from their natural higher plants [2-4], where this enzyme seems to be habitats or were grown in the Botanical Garden. universally distributed irrespective whether a plant Harvested samples were either directly freeze-dried produces amines or not [5]. The transaminase has or stored at - 18° until use. Polypodium vulgare was also been demonstrated in bacteria [6, 7], Enzymes collected in kg amounts in habitats near Altenahr producing aliphatic monoamines by amino acid (Rheinland) and Ameland (Holland). Fern gameto- decarboxylation are the bacterial valine decarbox­ phytes were grown on moist peat in a green house. ylase (EC 4.1.1.14) and the algal leucine decarbox­ ylase. Both enzymes have in common a rather broad Acetone pow der preparation substrate specificity. Besides valine and leucine about 7 to 10 related amino acids are decarbox­ Fresh or deep-frozen fronds (midveins removed) ylated. The bacterial enzyme was characterized from of Polypodium vulgäre were homogenized in 5 vol Proteus vulgaris [8] and Bacillus sphaerieus [9], the of acetone (—18°). Residue was collected by filtra­ algal enzyme from various species of Rhodophyceae tion under reduced pressure and extraction was (red algae) [10- 12], repeated twice in the same way. The resulting In the present communication we report on the powder was lyophilized, coarse particles were occurrence and partial purification of an amino acid removed by passage through a sieve (pore size 0.4 mm) and the final product was kept dry and Reprint requests to Prof. Dr. T. Hartmann. frozen at -18° until use: it can be stored without 0341-0382/84/0100-0024 $01.30/0 loss of activity for years. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. Th. Hartmann et al. • Aliphatic 1-Amino Acid Decarboxylase 25 Acetone powder could be further purified by Decar box) lase assay extensive extraction with 0.2 m phosphate buffer, Reactions were carried out in a Warburg pH 7.0 at 4° without considerable loss of de­ apparatus at 37 °C according to ref. [10]. Standard carboxylase activity. assay (total vol. 2.0 ml): 200-300 |imol K-phos- phate/Na-citrate at appropriate pH; 2|amol py- Enzyme extraction and partial purification ridoxal-5'-phosphate (PLP); 80(.tmol L-methio- nine (or alternative amino acid); 5 to 50 mg acetone Lyophilized fronds of Polypodium vulgare were powder or dry powder or 0.1- 1.0 ml soluble pulverized and passed through a sieve (pore size enzyme in reaction buffer. The reaction was started 0.4 mm). The dry powder can be stored at - 18°; by tipping in the substrate solution from the side- 5g were extracted in 100 ml buffer consisting of arme. 0.02 m phosphate buffer (pH 8.0), 0.35 m KC1, 0.1 m M diethyldithiocarbamate and 1 ml Triton- Amine estimation X-100. The suspension was stirred at 4° for 45 min. The residue was removed by centrifugation In some studies amino acid decarboxylation was (30 000 x # /15 min), the supernatant was referred to followed quantitatively by amine estimation accord­ as “crude extract”. ing to the method given in ref. [21]. The amine was This crude extract was made up 12.5% (w/v) with recovered from the reaction mixture by alkaline insoluble polyvinyl-pyrrolidone (Divergan-CE-5018, steam destination. Following reaction with BASF, Ludwigshafen) and stirred for 30 min. 2,4-dinitrofluorobenzene the resulting 2,4-dinitro- The precipitate was removed by centrifugation and phenyl derivatives were measured photometrically the supernatant was made up to 40% saturation with after TLC separation. The same chromatographic Butanol-1 at — 18° added drop by drop and conti­ procedure was applied for quantitative amine nuous stirring. The mixture was stirred for an addi­ separation and identification. tional 30 min, centrifuged (30 000 x g/ 15 min) and the aqueous layer was recovered. Residual butanol Proteine determination and low molecular compounds were removed by The protein was estimated by the Lowry method Sephadex-G-50 gel filtration. This step was [22]. combined with a buffer change to 50 m M T ris/H C l (pH 7.5) if a further purification by ion exchange chromatography was intended. Results The desalted enzyme solution was bound to a Distribution of the enzyme in ferns column (1.2x7 cm) of DEAE cellulose (Servacel DEAE-23-SH; 0.93 meq/g) equilibrated with 50 m M Using the sensitive amine assay acetone powder Tris/HCl (pH 7.5). The decarboxylase was eluted preparations of the following 27 fern species out of 9 families (taxonomy according to ref. [13]) were with a linear gradient of KC1 (0-0.6 m ), total vol. 200 ml. screened for their ability to decarboxylate leucine to 3-methylbutylamine: Aspidiaceae: Dryopteris borreri, D. carthusiana, Preparation of apoenzyme D. dilatata , D. filix-mas, D. villarii, Gymnocarpium Purified enzyme (10 ml. approx. lOOnkat) was dryopteris , G. robertianum, Polystichum aculeatum, mixed with 12.5 ml 0.4 m phosphate/citrate buffer P. braunii , P. lonchitis, P. setiferum. (pH 6.0) containing 1-methionine (final assay con­ Aspleniaceae: Asplenium ruta-muraria, A. trichoma- centration 60 m M ). The mixture was incubated at nes, A. viride, Phyllitis scolopendrium. 37 °C for 6 h, then placed in an ice-bath and pH was Athyriaceae: Athyrium distent ifolium, A. filix-femina, adjusted to 7.5. After centrifugation (19 000 x g / Cystopteris fragil is, C. montana, Mattheuccia 10 min) the supernatant was dialyzed against 0.01 m struthiopteris. Tris/H Cl buffer (pH 7.5) for 18 h. The dialysate Blechnaceae: Blechnum spicant. was centrifuged and the supernatant kept cool Hypolepidaceae: Pteridium aquilinum. (0-4°) until use. Ophioglossaceae: Botrychium lunaria. 26 Th. Hartmann et al. ■ Aliphatic 1-Amino Acid Decarboxylase Osmundaceae: Osmunda regal is. extracted at all. we succeeded to solubilize the Polypodiaceae: Polypodium vulgare. Polypodium enzyme at least partially from freeze- Thelypteridaceae: Thelypteris limbosperma, T. palus­ dried material in the presence of detergent at high tris. concentration (Table III). Triton-X-100 (1%) gave With the exception of two ( Athyrium distenti- the best results. Essentially no soluble activity could folium; Cystopteris montana) all species gave be detected when acetone powder was used instead positive results. Preliminary experiments revealed of dry material (Table III). Under the experimental that methionine and norvaline are most actively conditions applied approximately 50% of activity decarboxylated. With both substrates comparably high decarboxylase activities were determined monometrically for a variety of species (Table I). Table 11. Decarboxylase activity in the different fern generations. The fern sporophvlls contained decarboxylase ac­ tivity at all developmental stages. Phyllitis scolopen­ M ethionine drium sporophylls displayed the highest enzyme decarboxylation