JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, Suppl 5, 27–37 www.jpp.krakow.pl P. ROMER1, 3, A. FALTERMEIER1, V. MERTINS1, T. GEDRANGE2, R. MAI2, P. PROFF1 INVESTIGATIONS ABOUT N-AMINOPROPYL TRANSFERASES PROBABLY INVOLVED IN BIOMINERALIZATION 1Department of Orthodontics, Regensburg University, Germany; 2Department of Orthodontics, Preventive and Pediatric Dentistry, Ernst-Moritz-Arndt University Greifswald, Germany; 3Department of Biochemistry I, Regensburg University, Germany Polyamines are widespread distributed all over in living organisms. In Thalassiosira pseudonana 10 N-aminopropyl transferase like nucleotide sequences exists. It is assumed that these sequences are involved in the biomineralization of the diatom shell. The cDNA of the sequences were cloned, recombinant overexpressed and assayed with decarboxylated S-adenosylmethionine and several radioactive labelled polyamines. However, only a spermidine synthase and a thermospermine synthase were found to be enzymatically active in an in vitro assay. Both enzyme activities could be recognized in the crude extracts of Thalassiosira pseudonana and Cyclotella meneghiana. In further investigations the kinetics of the thermospermine synthase was determined and a site-specific mutagenesis of the bindig cavity of decarboxylated S-adenosylmethionine was carried out. Key words: biomineralization, N-aminopropyl transferase, polyamine, thermospermine INTRODUCTION Polyamines are cationic and amphiphilic organic molecules that are ubiquitously present in almost all known organisms. Common polyamines like putrescine, spermidine and spermine are involved in fundamental physiological and biochemical processes, i.e. in stress response, stabilization of DNA, the synthesis of the uncommon aminoacid hypusine and in the modulation of ion channel function (1-7). Some derivatives of polyamines were found in venoms of spiders (8). Other unusual long chain polyamines (pentamines, hexamines, quaternary and branched 28 polyamines) were found in thermophilic archaeas and bacterias and these are thought to be involved in preventing thermal degradation of nucleic acids (6, 9, 10). However, extensive work by the Sumper-Lab demonstrated that even psychrophilic diatoms contain long chain polyamines in the siliceous diatom cell wall (11, 12). These long chain polyamines are encased in the valve either in a unassociated form or in association with polycationic peptides, namely silaffins (12, 13). The main feature of diatoms is the siliceous cell wall that is composed of two overlapping valves. In vitro experiments by Kroger et al. have demonstrated, that silaffines and free long chain polyamines catalyze the polycondensation of monosilicic acid to polymeric silica (13). Moreover, Sumper et al. have suggested that long chain polyamines are presumably implicated in the nanopatterning of the siliceous cell wall by phase separation (14). The common way of polyamine synthesis is catalyzed by N-aminopropyl transferases. In this way, an N-aminopropyl moiety from decarboylated S-adenosylmethionine is transfered by an N-aminopropyl transferase to a polyamine acceptor molecule (15). However, the synthesis pathway of long chain polyamines is largely unknown. It was hypothesized by Sumper (personal communication) that the synthesis of long chain polyamines is likely mechanistically related to the formation of spermidine or spermine. The objective of the research presented in this paper was to examine this assumption. In this concept, we found an unusual N-aminopropyl transferase that is closely related to the ACL5-type of Arabidopsis thaliana (16) and that synthesizes only thermospermine instead of spermine by consumption of spermidine and decarboxylated S-adenosylmethionine (17). However, a detailed biochemical description of the thermospermine synthase has not been published. In the present study, we want to give a more detailed enzymological and biochemical insight into the thermospermine synthase and we want to present the discovery of a spermidine synthase from T. pseudonana. MATERIAL AND METHODS Cultures T. pseudonana clone CCMP1335 was cultivated in an artificial seawater medium as delineated by Poulsen and Kroger (18). Chemicals and molecular biological products [14C]-spermidine and [14C]-putrescine were purchased from GE Healthcare. S- adenosylmethionine was purchased from Fluka and decarboxylated according to Zappia et al. (19). All enzymes used for molecular biological techniques were provided by New England Biolabs. Synthesis of PCR primers and sequencing was carried out by MWG Biotech AG (Ebersberg, Germany). Cloning of N-aminopropyl transferases cDNA from T. pseudonana The purification of total RNA and the amplification and cloning of TPS_41289 cDNA was performed as published before (17, 20, 21). The TPS_108361 cDNA was amplified with Taq DNA- 29 polymerase using the forward primer 5´-ATGGCTGCCTTCGCCCCAC-3´ and the reverse primer 5´- CTACTTCGACAAACCACCG-3´. The PCR program was created by an initial denaturation step of 2 min at 95°C followed by 35 cycles for 20 s at 95°C, for 20 s at 58°C and for 60 s at 72°C. After PCR, 4 µl of the PCR reaction sample were directly cloned into the pQE-30 UA vector and subsequently transformed into E. coli SG13009 according to the manufacturer´s instructions (Qiagen). Recombinant protein purification For purification of the expressed enzymes, 100 ml of Luria-Bertani broth containing the antibiotics required for selection was inoculated with a single colony of the transformant. This culture was incubated at 37 °C at 160 rpm until the optical density at 600 nm reached 0.6. Isopropylthiogalactopyranoside (IPTG) was added (1 mM), and the culture was further incubated for 4 h at 37°C. Bacteria were harvested by centrifugation, suspended in 400 µl of 0.1 M Tris-HCl buffer pH 7.5 and ruptured by ultrasonication. After centrifugation at 20 000 × g for 10 min at 4°C, the crude extract was obtained. The crude extract was applied to a Ni-NTA-agarose column (Qiagen) and the 6×His-tag fusion-proteins were purified and dialyzed according to the instructions for native purification from the manufacturer. Enzyme assays To examine the N-aminopropyl transferase activity of TPS_108361 and TPS_41289, each pure recombinant protein was incubated in 100 mM Tris/HCl, pH 7.5 at 30° for 30 min with 2 nmol dcSAM and 23 pmol of [14C]-spermidine (5500 dpm) in a reaction volume of 150 µl. The reactions were stopped by heat inactivation (10 min, 95°C). The optimal temperature for recombinant TPS_41289 was examined by incubation at different temperatures ranging from 0°C to 65°C in 100 mM Tris/HCl, pH 7.5 with 5 µg protein for 15 min. The optimal pH of recombinant TPS_41289 was determined using different pH-buffers ranging from pH 6.8 to pH 10.6 with 5 µg protein at 55°C for 15 min. The kinetics of TPS_41289 were determined by varying the substrate concentration (0-0.36 mM dcSAM resp. 0-0.133 mM spermidine) in the reaction mixture under saturation of the another substrate (dcSAM: 0.24 mM resp. spermidine: 0.33 mM). Site-specific mutagenesis of TPS_41289 TPS_41289 were mutagenized by performing site-specific mutagenesis by overlap extension outlined by (20) in order to obtain D156 instead of E156. For the first amplification two separate PCR reactions were carried out using the primers: BA-VH1 (5´-CAAGAGCTCCTTCCGCTACG-3´), BA- VH2 (5´-CGAGCAGTGGCCAAGTCTC-3´) and BA-HH1 (5´-GAGACTTGGCCACTGCTCG-3´) and BA-HH2 (5´-GTCGTCATTCTCATCCTCAGG-3´). Both different amplificates were purified from the reaction vessel and subjected to a shared second PCR reaction using primers BA-VH1 and BA-HH2. The obtained PCR product was digested by restriction enzymes SphI and SacI and the resulting fragment was cloned into a pretreated pET-20b/41289-Vector. The further experiments were carried out as delineated in this paper. Sample preparation and thin-layer chromatography The reaction samples were vortexed with 1 vol methanol:chloroform (3:2) and centrifuged at room temperature for 2 min. The upper aqueous phase was collected in a fresh reaction tube and subjected to a heatable vacuum rotor to completely remove the aqueous phase. The polyamine samples were resuspended in 100 µl methanol and subsequently applied to a silica gel TLC-plate. The enzyme assays were analyzed by thin-layer chromatography (TLC) in the solvent system BAPF (butanol/acetic acid/pyridine/37% formaldehyde = 3/3/2/1) or in the BAPW (butanol/acetic acid/pyridine/water = 3/3/2/1) according to Mai et al. (21). The silica gel TLC-plates (Merck) were dried and exposed to a 30 phospho-imager screen (Fuji) for at least 2 h. The phospho-imager screen was analyzed on a Fuji BAS Reader 1000. The polyamine products were identified according to method published before (17, 21). Miscellaneous methods Analytical PAGE was carried out according to Sambrook et al. (22). For SDS-PAGE, 7.5% (wt/vol) stacking gels and 10 or 12% (wt/vol) running gels were used. Native PAGE was performed under protein preserving conditions employing 5% (wt/vol) stacking gels and 7.5% (wt/vol) running gels. Proteins were stained with Coomassie brilliant blue G-250. The protein quantification was achieved according to method described in previous studies (23). The participants gave their written informed consent and the study was approved by the local ethics committee. RESULTS Searching for N-aminopropyl transferase like sequences in a cDNA-library of T. pseudonana The genomic sequenze of the diatom is available on the website of the "Doe Joint Genome Institute" (www.jgi.doe.gov). The