Nucleoside Diphosphate Kinase of Halobacteria - Amino Acid Sequence and Salt-Response Pattern

գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ

Journal of Japanese Society for Extremophiles (2004), Vol. 3, 18-27 ଙཉ൫ۍ Mizuki T1, Kamekura M2, Ishibashi M3, Usami R1, Yoshida Y1, Tokunaga M3, Horikoshi K1

Nucleoside diphosphate kinase of halobacteria - Amino acid sequence and salt-response pattern -

1 Department of Applied Chemistry, Faculty of Engineering, Toyo University, Kawagoe, Saitama 350-8585, Japan 2 Noda Institute for Scientific Research, Noda, Chiba 278-0037, Japan 3 Department of Agricultural Chemistry, Kagoshima University, Kagoshima 890-0065, Japan Corresponding author: Kamekura M, [email protected] Phone: +81-4-7123-5573. Fax: +81-4-7123-5953.

Received: Oct. 15, 2003 / Accepted: Nov. 27, 2003 Abstract Nucleotide diphosphate kinase (NDK) was purified exchange g-phosphates between nucleoside triphosphates and from 12 strains of halobacteria using ATP-agarose diphosphates, thus playing a key role in maintaining cellular chromatography and their N-terminal amino acid sequences pools of all nucleoside triphosphates 1). NDK of halobacteria were determined. The electrophoretic mobilities of these was first studied as a major cytosolic protein of Natrialba enzymes differed significantly on the native-PAGE or the SDS- magadii (formerly Natronobacterium magadii) that bound to PAGE when the samples were not heat treated. Comparison of ATP-agarose and eluted with 5 mM ATP (adenosine seven complete amino acid sequences from seven species of triphosphate) in the presence of 3.5 M NaCl 16). This enzyme Haloarcula and those from Har. californiae and Har. japonica, was active over a wide range of NaCl concentrations; from whose N-terminal 3 amino acids have not been determined yet, 0.09 M to 3.5 M. Independently, Ishibashi et al. 5) purified revealed that they are very similar and differed at only 1 to 4 NDK from a strain of Halobacterium salinarum as a protein residues in 153 residues. NDKs from Haloarcula quadrata and that bound to ATP-agarose in the absence of salt and eluted Har. sinaiiensis differed only at the 30th amino acid (arginine with ATP. Further detailed experiments have demonstrated that vs. cysteine), yet they showed a remarkable difference in their NDK is one of the few halobacterial proteins that do not salt-response patterns, suggesting that a single amino acid require high salt concentrations to maintain their stability 6, 17). substitution can cause a one molar shift in the optimal NaCl An alignment of more than 100 published NDK amino acid concentration. sequences has shown clear conservation of primary structure throughout Eucarya, Bacteria, and Archaea (Kamekura, Key words extremely halophilic, Archaea, haloarchaea unpublished data). Although the NDK from the strain of Halobacterium salinarum is the only halobacterial NDK Introduction whose complete amino acid sequence has been published 5), Halobacteria are extremely halophilic Archaea that require three more sequences have been deduced from the genome high salt concentrations for growth. They are currently sequence of Halobacterium sp. NRC-1 14) and from two contig classified in 18 genera belonging to the family sequences publicly available, contig 131 of Haloarcula Halobacteriaceae 3, 10). The majority of strains grow best at marismortui and contig 3063 of Haloferax volcanii (consult NaCl concentrations of 3.5-4.5 M. To compensate for the high http://zdna2.umbi.umd.edu/~haloweb/). The sequence of the NaCl concentration in the environment, halobacteria NDK gene from Halobacterium sp. NRC-1 is identical to that accumulate intracellular KCl as high as 5 M. Most of the Hbt. salinarum strain 5). The three NDKs consist of 160 halobacterial proteins are adapted to function in the presence of amino acids (Hbt. salinarum) or 153 amino acids (Har. high salt concentrations and lose their activities at low salt marismortui and Hfx. volcanii) (see Results), and can be concentrations 12). aligned without gaps or insertions. The Hbt. salinarum NDK Nucleoside diphosphate kinase (NDK, EC: 2.7.4.6) has possesses one additional residue at N-terminus and 6 additional been shown to be almost ubiquitous in living organisms, with residues at C-terminus. The pair-wise similarities between the just a few exceptions 15). The main function of NDK is to three NDKs in the overlapping 153 residues are high, ranging 18 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ from 78 to 88 %. Given the close similarity of these NDKs, a debris, the supernatant was applied to a small column (0.7 ml comparison of their salt response patterns and stabilities may bed volume) of ATP-agarose (Sigma, 4% cross-linked, C-8) provide insights into the way these enzymes have adapted to equilibrated with SB. After washing four times with 1.5 ml function at different salt concentrations. aliquots of SB, NDK was eluted with 1.5 ml of the SB In this study, we a) purified NDKs from strains of containing 3 mM ATP, yielding homogeneous preparation (see halobacteria and investigated their electrophoretic behavior on Results), which was stored at - 30ò. polyacrylamide gels, b) determined N-terminal amino acid sequences and PCR-amplified gene sequences, allowing a Amino acid sequence determination detailed comparison of their protein sequences, c) studied the N-terminal amino acid sequences of the purified NDK salt response patterns of NDKs from some halobacteria. preparations were determined with an Applied Biosystems 492 Protein Sequencer. Forty five µl of the preparation was loaded Materials and methods on to a ProSorb membrane and washed with 500 µl of 0.1% trifluoroacetic acid to remove the Tris in the elution buffer. Cultivation of strains and purification of NDKs The following halobacterial strains were used in this study: Polyacrylamide gel electrophoreses Halobacterium sp. NRC-1 JCM 11081, Haloarcula Native and sodium dodecyl sulfate (SDS)-PAGE were vallismortisT ATCC 29715T, Har. aidinensis JCM 10024T, Har. performed using precast 15 -25 % polyacrylamide gels argentinensis JCM 9737T, “Har. californiae” ATCC 33799, (Daiichi Pure Chemicals, Tokyo) according to the methods Har. japonica JCM 7785T, Har. hispanica ATCC 33960T, Har. recommended by the manufacturer. For SDS-PAGE, 8 µl of marismortui JCM 8966T, Har. quadrata JCM 11048T, “Har. NDK preparation was mixed with 8 µl of a sample buffer sinaiiensis” ATCC 33800, Haloferax volcaniiT WFD11 (from containing 2% SDS, 30% glycerol, 0.125 M Tris-HCl, pH 6.8, Dr. WF Doolittle, Dalhousie Univ., Canada), 10 % 2-mercaptoethanol, and 0.01 % BPB. The mixture was Halogeometricum borinquenseT JCM 10706T, Halomicrobium either boiled for 3 min or not heat-treated before loading to the mukohataeiT JCM 9738T, Halorubrum saccharovorumT JCM gel. In some experiments the mixtures were heated at various 8865T, Haloterrigena sp. GSL-11 (from Dr. FJ Post, Utah State temperatures and for various periods. After electrophoresis Univ., USA), Natrialba asiaticaT JCM 9576T, Nab. magadii with a constant current of 40 mA at 5ò, the gels were stained NCIMB 2190T, Natrinema pellirubrumT JCM 10476T, and with Coomassie Brilliant Blue. Natronomonas pharaonisT JCM 8858T. Most of the neutrophilic strains were cultivated in 20 ml Extraction of genomic DNA, PCR amplification and (for the extraction of genomic DNA) or 200 ml (for the sequencing of NDK genes preparation of NDK) of a medium containing 1% casamino Halobacterial cells grown up in 20 ml medium were harvested, acids, 1% yeast extract, 0.1 % sodium glutamate_H2O, 0.3 % washed, and suspended in 25% NaCl, 10mM Tris-HCl, pH 7.6. trisodium citrate_2H2O, 0.2% KCl, 2% MgSO4_7H2O, and Cell lysis was accomplished by adding N-lauroyl sarcosine at a 20% NaCl, pH 6.5. Some strains required a medium with pH final concentration of 1 % (w/v), and DNA was purified as adjusted to 7.1. The alkaliphilic strains, Nab. magadii and Nmn. described previously 8). pharaonis were cultivated in a medium consisting of 1% NDK genes were amplified by PCR using Platinum Taq

Bacteriological Peptone (Oxoid), 0.3% trisodium citrate_2H2O, DNA Polymerase High Fidelity (Invitrogen). Amplification of

0.1% MgSO4_7H2O, 0.2% KCl, 22.5% NaCl, and 1.6 % the genes from Haloarcula strains was done with a primer set sodium carbonate. Incubation was at 37ºC in 150 ml or 500ml of a sense primer, 5’-GAG CGC ACG TTC GTG ATG GTC Erlenmeyer flask with orbital shaking at 150 strokes per AAI CCI GAC G (see Results) and an antisense primer-1, 5’- minute. GAG TTC GTC CAG TCG GTC GGC AAC CTT CTC A or The cells in a 200 ml culture were harvested at late an antisense primer-2, 5’-AGC CGT CGA GCT GGT AGA exponential phase, suspended in 10 to 15 ml, depending on the GCG TCA GGC TCA. The antisense primer-1 and 2 were cell density, of 3 M NaCl sonication buffer (SB) containing 2 synthesized from the sequence of nucleotide 138-108 and mM MgCl2 and 50mM Tris-HCl, pH 8.0 and disrupted by nucleotide 97-68 downstream from the stop codon of the ndk sonication with a Branson sonifier for 10 to 15 min on ice. gene of Har. marismortui delineated in the contig 131. The After spinning at 10,000 rpm for 20 min to get rid of cell expected lengths of amplified products were 591bp and 550bp, 19 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ respectively. The NDK reaction was started by adding 3 µl of the For the gene amplification, though partial, of strains of enzyme preparation, which had been diluted and kept on ice, other genera, an antisense primer-3, 5’-GTC CCA GTC CAC to the 97 µl of the NDK-RM and incubating at 37ò in a water GAG CTC IIC GTC GTC GAA GAA, was designed from the bath. After 1 min incubation, 1 µl aliquot was taken and two sequences (AB036344 and contig 131) encoding a injected into the 616 µl LL-RM to measure a relative relatively conserved amino acid sequence, FFDD(or E)D(or luminescence (RLU). After 11 min incubation, another 1 µl A)ELVDW(or Y)D(or E), corresponding to 135-145th amino aliquot was taken and RLU was determined. The increase of acids of Halobacterium sp. NRC-1 NDK. The expected length the RLU during the 10 min incubation was plotted against of amplified products was 423bp. The thermal cycling used the NaCl concentration of the NDK-RM. Calibration curves with following conditions: 25 cycles of 30 sec/94ò, 30 sec/55ò, known concentrations of ATP showed that 1 µM ATP gave 60 sec/72ò (for antisense primer-1 and 2) or 30 sec/72ò (for RLU of 215,000. One unit of the NDK is defined as the antisense primer-3). PCR products were blunted, cloned into amount of enzyme activity that produces 1 µmol of ATP in the SmaI site of pUC119, and transformed to Escherichia coli 1 min at 37 ò . In some experiments, the activity was JM109. Plasmids were purified from at least five transformants determined by a two-step assay. ADP produced in a reaction and both strands were sequenced with BcaBEST sequencing mixture with ATP as a phosphate donor and thymidine primers M13-20 and RV-M using a Beckman Coulter capillary diphosphate as an acceptor was determined by an enzyme- DNA sequencer CEQ2000XL. coupled method 5).

Measurement of NDK activities Results A 100 µl aliquot of the NDK preparation was dialyzed at 5ò in a Slide-A-Lyzer Dialysis Cassette (PIERCE) against 300 ml Protein purification and N-terminal amino acid sequencing of SB containing either 0.05 M or 3 M NaCl to remove the At the initial stage of our work before the contig sequences of 3 mM ATP in the sonication buffer. The buffer was exchanged Har. marismortui and Hfx. volcanii became available, we several times. Protein concentrations of the dialyzates were purified the NDKs from strains representing three genera of determined using BioRad protein assay solution with bovine halobacteria; Halobacterium sp. NRC-1, Haloarcula serum albumin as a standard protein. hispanica, and Haloferax volcanii. Preliminary data showed The NDK activity was measured using guanosine they differed significantly in the salt concentration -activity triphosphate (GTP) and adenosine diphosphate (ADP) as profiles and in the behavior on native-PAGE. Halobacterium substrates, and the amount of ATP produced was determined sp. NRC-1 and Hfx. volcanii always gave sharp bands with a via a luciferase assay 7) and read in a Lumitester K-210 relative motility of 0.54 and 0.19, respectively. The NDK of luminometer (Kikkoman Co.). The NDK-reaction mixture Har. hispanica, on the other hand, gave either a single, diffuse (NDK-RM), in a final volume of 100 µl, contained 1.0 mM band with a relative mobility of 0.2 to 0.3, or a mixture of the

GTP, 0.5 mM ADP, 25 mM MgCl2, 50mM Tris-HCl, pH 7.6 diffuse band with an additional sharp band having the same and NaCl (final concentrations of 0, 1, 2, 3, and 4 M). Ninety mobility as that of Hfx. volcanii NDK. These data prompted us seven µl of NDK-RM was added to each of a number of to expand the scope of investigation to strains of other genera, microtubes while the dialyzed NDK preparation, diluted as well as to several strains of a single genus. For the latter appropriately, was kept on ice until the reaction was started. aspect, members of the genus Haloarcula were chosen, as this The luciferin & luciferase reaction was done in a microtube genus contains as many as nine species (including invalid special to the Lumitester, to which 600 µl of a substrate mix species). We expected that the variation in the amino acid

(86 µ M D-luciferin, 5 mM MgCl2, and 25 mM Tris-HCl, pH sequences within the same genus might be lower than that 7.6) and 1 µl of NaCl solution (4, 3, 2, 1, and 0 M, to among genera, allowing the identification of residue(s) compensate for the NaCl in the NDK-RM) were added responsible for the differences in the behavior in PAGE and in beforehand. At 20 seconds before the luciferin & luciferase salt response pattern of the activities, if any. reaction, 15 µl of a luciferase solution (800 Mega LU, Twelve halobacterial strains belonging to six genera Kikkoman Co.) was added to make up the Luciferin listed in Table 1 yielded sufficient amounts of purified NDK Luciferase-Reaction Mixture (LL-RM), 616 µl in total, which for amino acid sequencing and polyacrylamide gel was kept at room temperature (24ò). electrophoresis. The N-terminal amino acid sequences of the 20 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ

NDK preparations suggested that the initiation methionine Thus, it was concluded that the NDKs of Halobacterium sp. residues were deleted post-translationally in all strains (Fig.1). NRC-1, Har. marismortui, and Hfx. volcanii are composed of The absence of the initiation methionine in the Har. 160, 153 and 153 amino acid residues, respectively. marismortui NDK was also confirmed in a separate experiment.

Table 1. Electrophoretic patterns of nucleoside diphosphate kinase from halobacteria ------SDS-PAGE Native-PAGE ------Strains >80ò, 3min 50ò, 3min not heated ------Haloarcula Vallismortis smear monomer smear smear argentinensis smear monomer smear smear quadrata smear monomer smear smear aidinensis trimer monomer smear smear sinaiiensis monomer + smear monomer monomer hispanica trimer monomer trimer ------Halobacterium sp. NRC-1 monomer monomer monomer monomer ------Haloferax volcanii WFD11 trimer monomer trimer trimer ------Natrialba asiatica smear monomer monomer smear magadii monomer monomer monomer ------Natrinema pellirubrum smear monomer smear smear ------Haloterrigena sp. GSL-11 smear monomer smear smear ------Trimer means a protein band that migrated to a position seemingly equivalent to a trimer in relative mobilities.

Hbt. sp. NRC-1 TDHDERTFVMVKPDGVQRGLIGDIVTRLET Har. aidinensis SEHERTFVMVKPDGVQRGLIGDIVSRFED Har. argentinensis SEHERTFVMVKPDGVQRGLIGDIVSRFED Har. hispanica SEHERTFVMVKPDGVQRGLIGDIVSRFED Har. quadrata SEHERTFVMVKPDGVQRGLIGDIVSRFED Har. sinaiiensis SEHERTFVMVKPDGVQRGLIGDIVSRFED Har. vallismortis SDHERTFVMVKPDGVQRGLIGDIVSRFED Hfx. volcanii SDAERTFVMVKPDGVQRGLIGDIVSRFED Htg. sp. GSL-11 SDHERTFVMVKPDAFARGLVGEEVSRLED Nab. asiatica GDHERTFVMVKPDAFARGLVGEXISR Nab. magadii SEHERTFVMVKPDAFARGLVGEEISRLLE Nnm. pellirubrum DHERTFVMVKPDAFARGLVGEEVDRLLD ********** ***.*. . * Fig. 1. N-terminal amino acid sequences of halobacterial NDKs. Asterisks indicate identical amino acids and dots indicate similar amino acids.

21 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ

12 3456789101112 Fig. 2. Polyacrylamide gel electrophoreses of NDK A preparations from 12 halobacterial strains. A: Native-PAGE, B: SDS-PAGE (boiled for 3 min), C: SDS-PAGE (not heated). Lane 1 Haloarcula vallismortis, Lane 2 Haloarcula argentinensis, Lane 3 Haloarcula quadrata, Lane 4 Haloarcula aidinensis, Lane 5 Haloarcula sinaiiensis, Lane 6 Haloarcula hispanica, Lane 7 Halobacterium sp. NRC-1, Lane 8 Haloferax volcanii, Lane 9 Natrialba asiatica, Lane 10 Natrialba magadii, Lane 11 Natrinema pellirubrum, Lane 12 Haloterrigena sp. GSL-11.

Behaviors of NDK proteins on polyacrylamide gel electrophoreses Native-PAGE of these preparations again revealed a variety of patterns (Fig.2A). NDKs from Halobacterium sp. 12 3456789 101112 NRC-1 and Natrialba magadii each gave relatively sharp B single bands with relative mobilities of about 0.5. SDS-PAGE of the preparations heat treated at 80ò or higher temperature for 3 min with SDS showed that all of them were almost completely homogeneous and they had almost the same molecular mass except for that of Halobacterium sp. NRC-1 (Fig. 2B). Figure 2C is an SDS-PAGE in which the samples were not heat-treated before loading to the gel. A separate run with a molecular marker indicated that the relative mobility of Hfx. volcanii NDK corresponded to a molecular mass of 56 KDa, which seemed to be a trimer of 17.4 KDa (calculated) monomers. Heating of Hfx. volcanii NDK with SDS at 50ò for up to 3 min gave a trimer, 70ò for 3 min gave a mixture of trimer and monomer bands, and 10 min heating resulted in a complete shifting to the monomer. On the other hand, heating of Haloterrigena sp. GSL-11 NDK with SDS at 70ò for 2 sec 12 3 4 5 6 7 8 9 10 1112 gave a diffuse band (smear), 30 sec heating gave a mixture of C smear and monomer, and 3 min heating resulted in a complete shift to the monomer. Table 1 summarizes the electrophoretic patterns of these preparations, demonstrating the considerable diversity in the NDKs from different genera, and among different species of the genus Haloarcula. It must be pointed out that the behaviors on the Native-PAGE and no heat- treatment SDS-PAGE differed slightly upon repeated experiments.

Determination of amino acid sequences of NDKs An alignment of sequences of franking regions of two NDK genes of Hbt. salinarum (AB036344) and Har. marismortui (contig 131, see Introduction) suggested that it was difficult to design PCR primers applicable to amplification of the 22 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ

complete NDK genes from strains of different genera (contig 3063 of Hfx. volcanii was not available at this point). Figure 1 shows that all halobacterial NDKs examined possess the conserved sequence, ERTFVMVKPDG(or A), corresponding to amino acids 5-15th of Halobacterium sp. NRC-1 NDK. The sense primer was, thus, designed based on the two gene sequences encoding the conserved amino acid sequence.

In order to amplify the genes of strains of the genus Haloarcula, the antisense primer-1 and 2 were synthesized as described in Materials and Methods. PCR with the sense primer and the antisense primer-1 yielded clear single bands with the expected size, 591bp, from eight strains of the genus Haloarcula except Har. aidinensis, which gave a very faint band, but replacement by antisense primer-2 yielded a clear band with the expected size, 550bp.

Figure 3 is the alignment of the amino acid sequences, which are combinations of the N-terminal sequences depicted in Fig. 1 and the deduced sequences from the amplified genes. Provided the N-terminal amino acids of Har. japonica and Har. californiae NDKs are the same as others, the 9 NDKs differed only at 1 to 4 residues each other, except that Har. quadrata and Har. californiae have the same sequence. The replacement of one or two amino acids observed in the four positions, 30th, 55th, 88th, and 138th depicted in Fig.3 have been ascertained by sequencing at least five clones. To confirm the replacement at 30th, 55th, and 88th positions (the138th position is included in the antisense primer-3), another PCR amplification done with a combination of the sense primer and the antisense primer-3, yielding exactly the same nucleotide sequences, confirming the replacements at the same positions. PCR amplifications of NDK genes, though partial, from strains of other genera were also done with a combination of the sense primer and the antisense primer-3 on type strains of type species of 12 genera, except for Hfx. volcanii. Pair wise Fig. 3. Alignment of amino acid sequences of NDKs from nine comparisons of the sequences (Fig. 4) revealed that similarities strains of the genus Haloarcula. Sequence of Har. marismortui among NDKs from different genera ranged from 67 to 84 %. was deduced from the contig 131 and N-terminal amino acid The following type strains failed to yield PCR products was determined experimentally. N-terminal amino acid probably owing to significant differences in the region of the sequences of Har. japonica and Har. Californiae NDKs have antisense primer: Natrinema pellirubrum, Natronobacterium not been determined yet. Shadow indicates identical amino gregoryi, Halobiforma haloterrestris, and Halorhabdus acids, except the second amino acid of Har. vallismortis NDK, utahensis. PCR on the type species of the following genera has D. Amino acids below the shadow are the consensus amino not been done yet: Halobaculum, Halococcus, Halosimplex, acids. Haloterrigena, and Natronococcus. The DDBJ/ GenBank/ EMBL accession numbers of the sequences determined in this 23 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ study are AB126337-AB126344, AB121062, and AB121066- AB121070.

Hbt. sp. NRC-1 TDHDERTFVMVKPDGVQRGLIGDIVTRLETKGLKMVGGKFMRIDEELAHE Har. Vallismortis SDHERTFVMVKPDGVQRGLIGDIVSRFEDRGLKMVGGKFMQIDQELAEE Hfx. Volcanii SDAERTFVMVKPDGVQRGLIGDIVSRFEDRGLKMVAGKFMQIDEELAHD Hgm. Borinquense VQRGLIGEIVSRFEDRGLKMVAGKFMQIDEELAHE Hmc. Mukohataei VQRGLIGDIVSRFEDRGLKLVGAKFMQIDRKLAEE Hrr. Saccharovorum VQRGLIGEIVSRFEERGLKLVGGKFMQIDEDLAHE Nab. Asiatica GDHERTFVMVKPDAFARGLVGEVISRLEDRGLKLVGIKVENMPRGRAEE Nmn. Pharaonis VQRGLIGDIVSRFEDRGLKLVAGKFMQIDDELARE ¥¥ ***.*....*¥* ***.* * . * .

Hbt. sp. NRC-1 HYAEHEDKPFFDGLVSFITSGPVFAMVWEGADATRQVRQLMGATDAQDAA Har. vallismortis HYGEHEDKPFFDGLVDFITSGPVFAMVWEGQDATRQVRTMMGETDPAESA Hfx. volcanii HYGEHEGKPFFEGLVDFITSGPVFAMVWEGQDATRQVRRMMGETDPAESA Hgm. borinquense HYGEHKDKPFFDGLVDFITSGPVFAMVWEGQDATRQVRRMMGETDPAESA Hmc. mukohataei HYGEHEGKPFFEGLVEFITSGPVFAMVWEGQDAVRQVRSMMGETDPAESA Hrr. Saccharovorum HYGEHEGKPFFDGLVDFITSGPVFAMVWEGADATRQVRAMVGETDPAESA Nab. asiatica HYSEHEDKPFYDDLVDFITGGPVVPMVWEGQDATRQVRQMIGETDPLEAA Nmn. pharaonis HYAEHVDKPFFDELKEFITSGPVFAMVWEGQDAVAQVRTMMGETDPAESA ** ** ***.. * *** ***¥ ***** ** *** ..* ** ..*

Hbt. sp. NRC-1 PGTIRGDYGNDLGHNLIHGSDHEDEGANEREIALFFDDDELVDWDRDASAWVYEDLADHD Har. Vallismortis PGTIRGDYGLDLGRNVIHGSDHEDEGANEREIELFFDEDELVDWDQIDAPWLYE Hfx. volcanii PGTIRGDYGLDLGRNVIHGSDHEDEGANEREIALFFDEDELVDYERVDETWLYE Hgm. borinquense PGTIRGDYGLDLGRNVIHGSDHEDEGANEREISL Hmc. mukohataei PGTIRGDFGLDLGRNVIHGSDHEDEGSNEREIEL Hrr. saccharovorum PGTIRGDFGLDLGHNVIHASDHEDEGANEREIDL Nab. asiatica PGTIRGDYALDLGRNVVHAADHEDEGANEREISI Nmn. pharaonis PGTIRGDFGLDLGRNVIHGSDTE-PGSAEREIGL *******. ***.*..* .* * *. **** .

Fig. 4. Alignment of amino acid sequences of NDKs from Halobacterium sp. NRC-1 and type species of seven genera. Sequences from Halobacterium sp. NRC-1 and Haloferax volcanii were deduced from the genome sequence 14), and contig 3063 (See http://zdna2.umbi.umd.edu/~haloweb/), respectively. N-terminal amino acids of NDKs from Haloarcula vallismortis and Natrialba asiatica were determined experimentally, as shown in Fig.1. Other sequences were deduced from PCR-amplified partial gene sequences, thus amino acids corresponding to the sense and antisense primers, ERTFVMVKPDG(or A) and FFDD(or E)D(or A)ELVDW(or Y)D(or E), respectively were not included. Abbreviations of generic names: Hgm., Halogeometricum; Hmc., Halomicrobium; Hrr., Halorubrum. Asterisks indicate identical amino acids and dots indicate similar amino acids.

Effect of NaCl concentrations on the NDK activities concentrations inhibited enzyme activity. The amino acid NDKs from Halobacterium sp. NRC-1, Hfx. volcanii WFD11, sequences of Halobacterium sp. NRC-1 and Hfx. volcanii and Haloterrigena sp. GSL-11 were dialyzed thoroughly NDKs were 78.4 % similar, in other words there were 33 against 0.05 M NaCl SB. Protein concentrations of dialyzates amino acids difference. were 16-26 µg/ml. Figure 5 A depicts the effects of NaCl Salt response patterns of other purified NDK concentration on the activities (the salt response patterns) of preparations (Table 1) were determined in this study. Although the enzymes. Halobacterium sp. NRC-1 and Haloterrigena sp. detailed discussions will be done elsewhere when the all of the GSL-11 NDKs exhibited essentially the same salt-response complete amino acid sequences become available, an pattern, with optimal activities at 1 M NaCl. The specific interesting difference in salt response pattern was found activity of the Halobacterium sp.NRC-1 NDK at 1 M NaCl between Har. quadrata and “Har. sinaiiensis” NDKs. The two was 60 U/ mg protein. On the other hand, Hfx. volcanii NDK NDKs differed only at the 30th amino acid; arginine in the showed a distinctly different pattern in that higher NaCl former and cysteine in the latter, as depicted in Fig. 3. The 24 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ former showed an optimal activity at 1 M NaCl, whereas the E. coli NDK could not be recovered by a similar ATP-agarose activity of the latter was highest at 2 M NaCl (Fig.5 B). chromatography in the presence of 0.05 M or 0.2 M NaCl. In the present study, the authors demonstrated that NDK proteins /.. A were purified to homogeneity from halobacterial strains 6. belonging to 6 genera, only with an ATP-agarose 4. chromatography even in the presence of 3 M NaCl. Although

2. we do not know the reason halobacterial NDKs are purified by

Pcj_rgtc_argtgrw&#' 0. a single ATP-agarose chromatography, Polosina et al., who

. purified the Natrialba magadii NDK in the presence of 3.5 M ./012 NaCl, suggested that “although the NDK is a minor protein, its L_Ajamlaclrp_rgml&K' strong affinity for ATP makes it a major component among the proteins that bind to ATP-agarose.” 16) NDK has been shown to be a multi-subunit enzyme B /.. composed of identical monomers 2). Eucaryotic NDKs (Human, 6. bovine retina, Drosophila melanogaster, Dictyostelium

4. discoideum) are hexamers, whereas bacterial enzymes (E. coli, Myxococcus xanthus) are reported as tetramers. The NDK from 2.

Pcj_rgtc_argtgrw&#' the hyperthermophilic archaeon Methanocaldococcus 0. jannaschii (formerly Methanoccoccus jannaschii) has been . overexpressed in E. coli and crystallized. Preliminary ./012 L_Ajamlaclrp_rgml&K' crystallographic data suggests it has a homohexameric structure 13), but its detailed enzymatic properties have not been reported. The NDK from Nab. magadii has been Fig. 5. Salt response patterns of halobacterial NDKs. A: suggested to be homooligomeric hexamer in the range of NaCl Halobacterium sp. NRC-1 (Ā), Haloferax volcanii (ć), and concentration from 0.45 to 3.5 M, by analytical Haloterrigena sp. GSL-11 ( ÿ ). 100% relative activities ultracentrifugation16). On the other hand, Halobacterium correspond to 550,886 RLU, 597,917 RLU, and 539,810 RLU, salinarum NDK, an equivalent of that of Halobacterium sp. respectively, measured in a Lumitester K-210. B: Haloarcula NRC-1, has been shown to change its subunit assembly sinaiiensis (Ā) and Haloarcula quadrata (ÿ). 100% relative depending on the concentration of NaCl, from dimeric at 0.2 M activities correspond to 580,972 RLU and 372,173 RLU, to hexameric at 3.8 M, as determined by the sedimentation respectively equilibrium method 6). In this context, the results of the native- and SDS-PAGE of Halobacterium sp. NRC-1 and Natrialba Discussion magadii NDK are of interest but also puzzling. Relative As of November 2003, NDK sequences from 153 organisms mobilities of Halobacterium sp. NRC-1 NDK in native-PAGE, have been registered in KEGG (Kyoto Encyclopedia of Genes SDS-PAGE (not heated), and SDS-PAGE (boiled) were exactly and Genomes, http://www.genome.ad.jp/kegg/kegg2.html), 16 the same (Fig.2 A, B, C). There exist preliminary data of which are archaeal. An alignment of more than 100 suggesting that the NDK stays dimeric even in the absence of sequences revealed a fairly well conserved primary structure NaCl (Ishibashi et al., unpublished data). This data may throughout the three domains, Eucarya, Bacteria, and Archaea. suggest that the NDK migrated as a dimer in the native-PAGE. We also noticed some signature amino acids residues specific Behaviors of other NDKs are also puzzling, including the to Eucarya (Kamekura, unpublished data). diffuse band (smear) to monomer and trimer to monomer Ishibashi et al. have shown that Halobacterium conversion upon heat treatment with SDS, but we need more salinarum NDK was separated from molecular chaperone information on the three dimensional structures of these DnaK, another major ATP binding protein by affinity column proteins in the high and low NaCl concentrations. chromatography using ATP-agarose column in the absence of Though various halophilic enzymes are supposed to NaCl 5). The same group also purified NDK from a moderately exhibit halophilic properties by different mechanisms, halophilic eubacteria Halomonas sp. in the presence of 0.2 M comparative analysis of amino acid sequence of enzymes NaCl 18). Our preliminary experiments showed, however, that 25 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ combined with mutational analyses is a powerful approach for structure models of Har. sinaiiensis and Har. quadrata NDKs analyzing the relative importance of certain residues in protein to see how the substitution of arginine by cysteine affects the stabilities and halophilicity. For example, malate overall structures. dehydrogenase (MDH) from Haloarcula marismortui is one of the most thoroughly investigated halobacterial enzymes 4). Acknowledgement The authors express sincere gratitude to Some time ago, Madern et al.11) showed that a mutant MDH Dr. Shiladitya DasSarma of University of Maryland protein with single amino acid change (Glu243Arg) was more Biotechnology Institute for providing unpublished contig halophilic, and required significantly higher concentrations of sequence data of two halobacteria, Haloarcula marismortui NaCl or KCl for equivalent stability. In another example, the and Haloferax volcanii. They also appreciate a critical reading halophilic serine proteinase halolysin R4, Kamekura et al.9) of the manuscript by Dr. Mike Dyall-Smith of University of showed that replacement of two cysteine residues by two Melbourne, Australia, and David Walsh of Dalhousie serine residues in the C-terminal extension decreased the University, Canada. stability of the enzyme, and the mutant Halolysin R4 also required significantly higher concentrations of NaCl for References optimum activity. 1) Bernard M.A., Ray N.B., Olcott M.C., Hendricks S.P., However, these two examples are perhaps not the most Mathews C.K. 2000. Metabolic functions of microbial useful representatives for studying the relative importance of nucleoside diphosphate kinases. J. Bioenerg. Biomembr. specific residues on the activity and stability of halophilic 32: 259-267. enzymes as they lose activity below 1 M NaCl. In another 2) Giartosio A., Erent M., Cervoni L., Morera S., Janin J., study, we have found that ureases from Haloarcula spp. also Konrad M., Lascu I. 1996. Thermal stability of hexameric lose activity at low salt concentration (Mizuki et al., Biosci. and tetrameric nucleoside diphosphate kinases. Effect of Biotechnol. Biochem., in press). The ideal protein for the study subunit interaction. J. Biol. Chem. 271: 17845-17851. of halophilicity should be an enzyme that is stable in the 3) Grant W.D., Kamekura M., McGenity T.J., Ventosa A. absence of salt and exhibits highest activity in the presence of 2001. Class Halobacteria class. nov. Order salt. For example, genes of ideal protein could be expressed in Halobacteriales. In: Boone D.R., Castenholz R.W., Escherichia coli to readily access large quantities of pure and Garrity G.M., (eds) Bergey´s manual of systematic active halophilic enzymes 5). In this sense, the NDK proteins bacteriology, 2nd ed, vol. 1. Springer-Verlag, New York, from Haloarcula spp. are quite promising in the future pp 294-334. investigation of halophilicity at the single-amino acid- 4) Irimia A., Ebel C., Madern D., Richard S.B., Cosenza substitution level. L.W., Zaccai G., Vellieux F.M.D. 2003. The oligomeric The difference in the salt response patterns of the Har. states of Haloarcula marismortui malate dehydrogenase quadrata and Har. sinaiiensis NDKs is quite interesting. The are modulated by solvent components as shown by 30th amino acid was arginine in Har. quadarata and cysteine crystallographic and biochemical studies. J. Mol. Biol. in Har. sinaiiensis. The arginine at this position is conserved in 326: 859-873. all halobacterial NDKs except the one of Har. sinaiiensis and 5) Ishibashi M., Tokunaga H., Hiratsuka K., Yonezawa Y., that of Halobacterium sp. NRC-1, which is substituted by Tsurumaru H., Arakawa T., Tokunaga M. 2001. NaCl- another basic amino acid, lysine. In almost all of the archaeal activated nucleoside diphosphate kinase from extremely and eucaryal NDKs, this position is occupied by arginine or halophilic archaeon, Halobacterium salinarum, maintains lysine, whereas in bacterial NDKs it is lysine or alanine. In this native conformation without salt. FEBS Lett. 493:134- sense the cysteine in this position is quite extraordinary. 138. Although the survey of the relationships between 6) Ishibashi M., Arakawa T., Philo J.S., Sakashita K., primary structure and salt-response pattern of halobacterial Yonezawa Y., Tokunaga H., Tokunaga M. 2002. NDKs has just begun, our present data suggest that a single- Secondary and quaternary structural transition of the amino acid-substitution may cause a drastic shift in the optimal halophilic archaeon nucleoside diphosphate kinase under salt concentration of halobacterial enzymes by as much as 1 high- and low-salt conditions. FEMS Microbiol. Lett. molar. Since X-ray structures of some NDKs have been 216: 235-241. determined, it will be possible to construct three dimensional 7) Kajiyama N. and Nakano E. 1993. Thermostabilization of 26 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ

firefly luciferase by a single amino acid substitution at 14) Ng W.V., Kennedy S.P., Mahairas G.G., Berquist B., Pan position 217. Biochemistry 32: 13795-13799. M., Shukla H.D., Lasky S.R., Baliga N.S., Thorsson V., 8) Kamekura M., Seno Y., Holmes M.L., Dyall-Smith M.L. Sbrogna J., Swartzell S., Weir D., Hall J., Dahl T.A., Welti 1992. Molecular cloning and sequencing of the gene for a R., Goo Y.A., Leithauser B., Keller K., Cruz R., Danson halophilic alkaline serine protease (Halolysin) from an M.J., Hough D.W., Maddocks D.G., Jablonski P.E., Krebs unidentified halophilic archaea strain (172P1) and M.P., Angevine C.M., Dale H., Isenbarger T.A., Peck R.F., expression of the gene in Haloferax volcanii. J. Bacteriol. Pohlschroder M., Spudich J.L., Jung K.W., Alam M., 174: 736-742. Freitas T., Hou S., Daniels C.J., Dennis P.P., Omer A.D., 9) Kamekura M., Seno Y., Dyall-Smith M.L. 1996. Ebhardt H., Lowe T.M., Liang P., Riley M., Hood L., Halolysin R4, a serine protease from the halophilic DasSarma S. 2000. Genome sequence of Halobacterium archaeon Haloferax mediterranei; gene cloning, species NRC-1. Proc. Natl. Acad. Sci. USA, 97: 12176- expression, and structural studies. Biochim. Biophys. Acta. 12181. 1294:159-167. 15) Pollack J.D., Myers M.A., Dandekar T., Herrmann R. 10) Kamekura M., Mizuki T., Usami R., Yoshida Y., 2002. Suspected utility of enzymes with multiple Horikoshi K., Vreeland R.H. 2003. The potential utility of activities in the small genome Mycoplasma species: the signature bases in the 16S rRNA gene sequences to aid in replacement of the missing “household” nucleoside the definition of genera of halobacteria. In: Ventosa A. diphosphate kinase gene and activity by glycolytic (ed) Halophilic Microorganisms. Springer-Verlag, pp.77- kinases OMICS 6: 247-258. 87. 16) Polosina Y.Y., Jarrell K.F., Fedorov O.V., Kostyukova A.S. 11) Madern D., Pfister C., Zaccai G. 1995. Mutation at a 1998. Nucleoside diphosphate kinase from single amino acid enhances the halophilic behaviour of haloalkaliphilic archaeon Natronobacterium magadii: malate dehydrogenase from Haloarcula marismortui in purification and characterization. Extremophiles 2:333- physiological salts. Eur. J. Biochem. 230: 1088-1095. 338. 12) Madern D., Ebel C., Zaccai G. 2000. Halophilic 17) Polosina Y.Y., Zamyatkin D.F., Kostyukova A.S., adaptation of enzymes. Extremophiles 4: 91-98. Filimonov V.V., Fedorov O.V. 2002. Stability of Natrialba 13) Min K., Song H.K., Chang C., Lee J.Y., Eom S.H., Kim magadii NDP kinase: comparisons with other halophilic K.K., Yu Y.G., Suh S.W. 2000. Nucleoside diphosphate proteins. Extremophiles 6:135-142. kinase from the hyperthermophilic archaeon 18) Yonezawa, Y., Tokunaga, H., Ishibashi, M., and Tokunaga, Methanococcus jannaschii: overexpression, M. 2001. Characterization of nucleoside diphosphate crystallization and preliminary X-ray crystallographic kinase from a moderately halophilic eubacteria. Biosci. analysis. Acta. Cryst. Section D 56:1485-1487. Biotechnol. Biochem. 65:2343-2346.

27