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 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 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 and a thermospermine synthase were found to be enzymatically active in an in vitro assay. Both 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 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 genome infomation revealed the existence of a number of putative N-aminopropyl transferase like sequences. These sequences were designated TPS_11130, TPS_108361, TPS_41289, TPS_41806, TPS_106272, TPS_22645, TPS_35489, TPS_105825 and TPS_23043. In order to examine the transcription of these putative N-aminopropyl transferase sequences, a reverse transcription-PCR (RT-PCR) was carried out. The PCR- analysis validated the transcription of TPS_11130, TPS_108361, TPS_41289, TPS_41806, TPS_106272, TPS_22645, TPS_35489 (depicted in Fig. 1), but not of TPS_105825 and TPS_23043 (not shown). Hence, it seems that TPS_105825 and TPS_23043 were not transcribed under culture conditions.

N-aminopropyl transferase assay with crude extracts of several diatoms To examine the assumption that the synthesis of long chain polyamines are mechanistically closely related to that of spermidine or spermine, dcSAM and

Fig. 1. Verification of transcription of putative N-aminopropyl transferase sequences by RT-PCR with subsequent agarose gel analysis (1,2 % w/v): (a) TPS_108361, (b) TPS_41289, (c) TPS_101424, (d) TPS_106272, (e) TPS_41806, (f) TPS_11130, (g) TPS_22645, (h) TPS_35495, (i) positive control: β-Actin. 31

[14C]-putrescine and [14C]-spermidine was incubated with several crude extracts of Cyclotella meneghiana and T. pseudonana which both belongs to the family of Thalassiosiraceae. A spermidine synthase activity was detectable in all diatome crude extracts (data not shown). The incubation of crude extract of T. pseudonana and C. meneghiana with [14C]-spermidine and dcSAM resulted in the detection of thermospermine (depicted in Fig. 2). However, the formation of longer polyamines were not observed.

Identification of two N-aminopropyl transferases In order to clarify the results of the enzyme tests in the crude extract of T. pseudonana, the homologues sequences of spermidine synthase and spermine

Fig. 2. Detection of spermidine-N- aminopropyl transferase activity after incubation of crude extracts of C. meneghiana and T. pseudonana with spermidine and dcSAM. The TLC were carried out by using the BAPW solvent system.

Fig. 3. Phylogenetic tree of N-aminopropyl transferases (created with http://align.genome.jp/). The distance scale reflects the evolutionary distance. Medicago truncatula (ABE91714), Arabidopsis thaliana (BAB83652), Malus sylvestris (BAE19760/ BAE19758), Thermus thermophilus (YP_004447), Saccharomyces cerevisisae (AAC17191), Caenorhabditis elegans (CAC37332). TPS: Thalassiosira pseudonana. 32 synthase were selected. Moreover, TPS_108361 displays a high sequence identity to other spermidine synthases or spermine synthases belonging to the group of SPDS/SPMS-like N-aminopropyl transferases. On the other hand, TPS_41289 shares a high sequence identity to other ACL5-like spermidine N-aminopropyl transferases (compare with Fig. 3).

A. Cloning, purification and enzyme assay of a spermidine synthase The protein coding cDNA of TPS_108361 was cloned into the expression vector pQE-30 UA. The induction with 1 mM IPTG at 37°C for 4h resulted in an overexpressed recombinant N-terminal hexahistidine fusionprotein. TPS_108361

Fig. 4. Coomassie-stained SDS-PAGE of purified recombinant (a) TPS_41289 and (b) TPS_108361 and (c) Native PAGE of TPS_41289.

Fig. 5. Interpretation of a TLC analysis (solvent sytem: BAPW) of an N- aminopropyl transferase assay for N- aminopropyl transferase activity: (a) putrescine (standard), (b) spermidine (standard), (c) incubation of dcSAM and spermidine with TPS_41289 resulted in thermospermine, (c) incubation of dcSAM and putrescine with TPS_108361 resulted in the formation of spermidine. 33 was purified by Ni-NTA-Agarose with a yield of 10 mg protein per 1L LB- Medium. The purity and the monomeric molecular weight of TPS_108361 was proved by SDS-Gele electrophoresis. A monomeric molecular weight of approximately 37 kDa was determined by SDS-PAGE (Fig. 4b). The incubation of pure TPS_108361 with dcSAM and putrescin in an in vitro assay led to the total conversion of putrescin to spermidin (Fig. 5). Furthermore, under protein saturating conditions TPS_108361 catalyzes the formation of spermidine to spermine to a smaller extent (data not shown). However, a N- aminpropyltransferase activity in presence of [14C]-1,3-diaminopropane and dcSAM was not observed.

B. Biochemical analysis of a thermospermine synthase The cloning of TPS_41289 and its recombinant overexpression was published by Knott et al. (17). The purified TPS_41289 possesses a hexahistidine tag at the C-Terminus. The protein was purified with Ni-NTA- Agarose and subsequently analyzed by SDS-PAGE and native PAGE. The monomeric molecular weight of TPS_41289 estimated by SDS-PAGE is appromately 48 kDa (Fig. 4a), which is in agreement with the known amino acid sequence. The analysis by Native PAGE resulted in an apparent molecular weight of 196 kDa (Fig. 4c). The pure recombinant protein was subjected to an enzyme test with dcSAM and spermidine at different temperatures and pH conditions. Interestingly, the highest enzyme activity was observed at 55°C. The optimal pH range was found to be at pH 9.4 to pH 9.6. A specific enzyme activity of 313 nmol per mg protein per minute was measured under the optimized reaction conditions. The KM of dcSAM was measured to be 0.091 mM, whereas the KM of spermidine was determined to be 0,104 mM. The incubation of pure recombinant TPS_41289 with dcSAM and several potential [14C]-polyamine acceptors (1,3-diaminopropane, putrescine, spermidine) clearly demonstrated that TPS_41289 catalyzes only the formation of thermospermine by the consumption of dcSAM and spermidine (Fig. 5).

Fig. 6. Autoradiogram of a TLC-analysis (solvent sytem: BAPF) after an enzyme assay using spermidine, dcSAM with (a) void enzyme, (b) TPS_41289 with changed D156 instead of E156, (c) TPS_41289 with unchanged E156 (control). 34

Changing of E156 to D156 in TPS_41289 and its impact on product formation A point mutation was introduced in TPS_41289 to obtain D156 instead of E156 in the amino acid sequence of the protein. The mutagenized TPS_41289 was cloned into the same expression vector as performed by Knott et al. (17). After IPTG induction and subsequent affinity purification the TPS_41289 was subjected to an enzyme test with dcSAM and spermidine. However, in comparison to unaltered TPS_41289 the modified TPS_41289 with D156 was able to synthesize, beside the main product thermospermine, even tiny amounts of spermine (Fig. 6).

DISCUSSION

Due to the significance of polyamines in several processes (biomineralization, cell proliferation, stress response) N-aminopropyl transferases are in the focus of interest of biochemists. Beside the interest in fundamental research, the N- aminopropyl transferases of Helicobacter pylori and several protozoes are regarded to be a promising target to compromise severe infections (24 - 27). The most known N-aminopropyl transferases contains a charaterisitic amino acid motif, such as "GGGD/EG", that is common for enzymes using dcSAM (17, 28). Almost all known organisms contain only one or two N-aminopropyl transferases (spermidine and ) that are responsible for the synthesis of polyamines. Surprisingly, T. pseudonana contains 10 N-aminopropyl transferase like sequences that have a more or less related sequence motif. However, from these sequences 8 N-aminopropyl transferases were definitivly transcripted under culture conditions. Moreover, only 2 N-aminopropyl transferases (TPS_108361 and TPS_41289) were found to be enzymatically active, whereas the others were not (data not published). Hence, the metabolic function of the sequences TPS_11130, TPS_22645, TPS_35495, TPS_41806, TPS_106272 and TPS_101424 remain to be elucidated. To our knowledge, TPS_108361 is the first published spermidine synthase from diatom origin. The amino acid composition is in terms of the sequence length and identity similar to other known spermidine synthases. Even the binding motif for dcSAM is composed in the same manner like those of the SPDS/SPMS-Group (spermidine or spermine synthase like sequences). In contrast to the spermidine synthase from Thermotoga maritima (28), TPS_108361 was not able to catalyze the formation of norspermidine in the presence of 1,3-diaminopropane and dcSAM. Other spermidine synthases like those of Thermotoga maritima, Plasmodium falciparum have been reported to be able to catalyze spermine by consumption of spermidine and dcSAM in a small extent (28, 29). Also TPS_108361 synthesizes spermine, but not with the same activity as with putrescine and dcSAM. Despite of the existence of numerous N- 35 aminpropyltransferase like sequences, TPS_108361 seems to be the only spermidine synthase in T. pseudonana. The cloning, purification and the analysis of the reaction products of TPS_41289 were readily published by Knott et al. (17). However, a more detailed characterization in terms of enzymology was omitted. The molecular weight of TPS_41289 was determined to be 210 kDa by size-exclusion chromatography (17). As a consequence, a homotetrameric composition of the protein was concluded. These result was verified by Native PAGE, whereby a apparent molecular weight of 196 kDa was found out. The highest enzyme activity at 55°C was determined for TPS_41289, whereas at physiological growth temperatures the enzyme activity was five fold less. This observation can be explained by the assumption that the enzyme activity of TPS_41289 is sufficient for the physiological function in the diatom. In addition, it is unclear whether the enzyme activity of the in vitro test is the same as in vivo. The measured optimal pH-range and the determined specific enzyme activity is in the same range with other investigated spermidine-N-aminopropyl transferases (30). TPS_41289 was very specific to its substrate spermidine but not for other polyamines, like 1,3-diaminopropane or putrescine. This observation is in accordance with results obtained by investigations about the substrate specifity of another spermidine-N-aminopropyl transferase (30, 31). This results probably from the fact that the hydrophobic polyamine binding pocket of N-aminopropyl transferases is the key feature for selecting polyamine substrates with the appropriate number of CH2 modules (28). The incubation of putrescine, spermidine and dcSAM in crude extracts of diatoms belonging to Thalassiosiraceae displays only the formation of spermidine resp. themospermine. This is in accordance to the enzyme assays. However, in the shells of T. pseudonana long chain polyamines were found, whereby the question arises by which way the long chain polyamines emerges. However, it cannot rule out that TPS_41289 is not involved in the formation of diatom specific long chain polyamines. The unavailability of radioactive labelled norspermidine, norspermine and other pentamines makes it impossible to analyze the product formation using the methode published by Mai et al. (21). Therefore, new technical approaches are now underway to solve this problem and in order to study the substrate affinity of TPS_41289 for longer polyamines that are non-radioactive. The main feature of N-aminopropyl transferases belonging to the ACL5- Group is the glutamate (E) instead of the aspartate (D) in the dcSAM binding motif (17). The introduction of a point mutation led, beside the main product thermospermine, to the production of small amounts of spermine. We assume that this changed amino acid results in a certain structural impact that in a unknown way favors the formation of two isomers of tetramines. It is more likely that other amino acids that interact with the amino groups of the polyamine substrate are the 36 key factors of product binding that determines the N-aminopropylization either of the 1N-position or 10N-position of spermidine.

Acknowledgement: Authors acknowledge the support of Manfred Sumper to perform of this work.

Conflicts of interest statement: None declared.

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R e c e i v e d : July 30, 2008 Accepted: October 15, 2008

Author’s address: Prof. Dr. P. Proff, Department of Orthodontics, Regensburg University, Franz- Josef-Strauss-Allee 11, D-93053 Regensburg, Germany; e-mail: [email protected]