Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves

304

Biochimica et Biophysica Acta, 1171 (19931304-306 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90435

Short Sequence-Paper

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves Jianing Zhang a, Toshiko Nomura ~, Kimio Yatsunami b, Akiko Honda ~', Yukihiko Sugimoto a, Tamako Moriwaki ", Jun Y a m a m o t o ~, Mitsuhiko Ohta c, Tetsuya Fukui a and Atsushi Ichikawa " " Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto Unil'ersity, Kyoto (Japan), h Japan Tobacco Inc., Pharmaceutical Basic Research Laboratories, Kanagawa (Japan) and '" Clinical Research Center, Utano National Hospital, Kyoto (Japan) (Received 31 August 1992)

Key words: Nucleoside diphosphate kinase; Spinach; cDNA sequence

The primary structure of nucleoside diphosphate (NDP) kinasc II, one of thc two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26 107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases a and /3, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6.) is a ubiquitous enzyme that catalyzes the transfer of the terminal phosphate group of 5'-triphosphate nucleotides to 5'-diphosphate nucleotides [1]. This enzyme has emerged recently as having a key role in fundamental cellular processes, based on the recent proposal that Nm23 and awd proteins were NDP kinase, on the basis of sequence homologies [2]. This proposal has been confirmed at the protein level for both Nm23 [3] and awd [4] proteins. In addition, the presence of a second isoform has been predicted for the rat N D P kinases [5] and human Nm23 protein [6]. This has also been demonstrated at the protein level for the Nm23 protein [3]. However, the functional difference between these two isoforms is unknown at present. Thus, much attention has been focussed on the other possible function(s) of these two NDP kinases. Meanwhile, N D P kinase is also known to be

Correspondence to: A. Ichikawa, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan. The sequence data reported in this paper have been submitted to the D D B J / E M B L / G e n B a n k Nucleotide Sequence Database under the accession number Dl1465. Abbreviations: NDP, nucleoside diphosphate; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography.

present in plant cells, however, this enzyme of higher plants has not been extensively characterized. Recently, we purified two types of spinach N D P kinase with molecular masses of 16 (NDP kinase I) and 18 kDa (NDP kinase II) to electrophoretic homogeneity [7]. The finding of two NDP kinase isozymes in spinach raises the possibility that NDP kinase may also have a regulatory function, such as signal transduction, in higher plants. In order to examine this issue, it is essential to know the primary structures of spinach N D P kinases and to compare them with those of other NDP kinases. The primary structure of N D P kinase I has been deduced from its c D N A sequence [8]. Here we present the nucleotide and deduced amino acid sequences of spinach N D P kinase II. Purified N D P kinase II [7] was digested with lysyl endopeptidase and the resulting peptide fragments were purified on reverse phase H P L C Cls or C 4 columns. The amino acid sequences of the purified peptides were then analyzed using a gas-phase peptide sequencer (Applied Biosystems, model 477A). A c D N A library derived from spinach leaves was constructed using the a g t l l vector [9]. The library was screened with a c D N A probe which was synthesized by amplifying a segment (nucleotides 703-940) of N D P kinase II using two primers (nucleotides 703-719 and oligo(dT)) by polymerase chain reaction (PCR). The nucleotide

305 sequence of the former primer had been derived from a fragment (nucleotides 343-940) of NDP kinase II, which had been obtained by PCR with two primers (a 36-mer containing all possible codons specifying amino acids K-L-I-G-L-K-M-Y-P-C-P-K-E and oligo(dT), see Fig. 1). The region encompassing nucleotides 703-719 of NDP kinase II exhibits only weak homology to the corresponding region in spinach NDP kinase I. It was confirmed that two clones, AN-IIa and IIb, with 1.0 and 1.1 kb cDNA inserts, respectively, contained the amino acid coding region (699 base pairs) of spinach NDP kinase II. NDP kinase II consists of 233 amino acid residues and has a molecular mass of 26 106.70 Da (Fig. 1). The N-terminal amino acid sequence of the purified spinach NDP kinase II was determined to be S-M-EQ-V-E-E-T-Y-I-M-I-K-P-D-G-V-Q-R-G-L-V-G-E-I-I-

GTTTGGTGAGAGAGAGAGA

-

1

ATGGAAGCAATGAGTGGATTGAGCAGCCCTTGCAATTGCATTTCATCACTACCTCACTCC

60

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240 80

TCCATGGAGCAAGTTGAGGAGACTTACATTATGATTAAACCAGATGGGGTTCAACGTGGC

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ATGTATCCATGCCCTA~GAATTAGCTGAGGAGCACTACAAGGATCTCAAGGCCk~AGTCA M Y P C P K E L A E E H Y K D L K A K S

360 120

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GGTG TTGGAGTTGTTGCTTCATCACGTAAGCTAATAGGAGCCACCGATCCTCTTCAAGCT G V G V V A S S R K L I G A T D P L Q A

540 280

GAACCTGGAACTATAAGAGGGGATCTTGCTGTTCA~CGGGGAGGAATGTGGTTCATGGA E P G T I R G D L A V Q T G R N V V H G

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AGTGATAGCCCTGACAATGGCAAGCGTGAAATAGGTCTGTGGTTTAAAGAAGGTGA~TA S D S P D N G K R E I G L W F K E G E I

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TGC~AATGGACACCTGCCCAGGCACCATGGCTAAGAGAGTGATCTTGTGACCCGTTATTT C Q W T P A Q A P W L R E *

720 233

TGTGAATCTACTGATAGAGTGTTGCACAGCTTTGAGGCTTTATTTGTTCTGGCTTGGGAC

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ATGCATTTTTCCGTGTGCTTCAAATGTACTTATTCTGTAGCAAAAATTTCTGTAGGATGT

840

GTATTTATCTACATTCACAATCACAAGTTTTTGAATCAATTACAAGTTGCTGCTGAAATA

900

~r

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TCCTGGTaATT/~,,~CATT~JOa~'.~

Fig. 1. c D N A and amino acid sequences of N D P kinase 11. In order to avoid a cloning artifact, D N A sequences of both strands of two overlapping clones (AN-I]a and llb) have been determined. The N-terminal amino acid sequence of the purified N D P kinase II (------) and the amino acid sequences of peptide fragments obtained by proteolytic digestion ( ) are underlined.

S - R - F - E - K - K - G - F - K - L - I - G - L - K - M - Y - P , which matched amino acid residues 81-123 of the cDNA-derived amino acid sequence determined in this study. The sequences of the seven peptide fragments obtained by proteolytic digestion were included in it (Fig. 1). Furthermore, it was found that the molecular mass of amino acid residues 81-233 was 17 083.37 Da, which is in agreement with that of the purified enzyme (18 kDa). Therefore, it is possible that the ATG codon at nucleotides 1-3 is the initiation codon and that the N-terminal amino acid residues (1-80) are post-translationally processed. Since the N-terminal region of the purified spinach NDP kinase I could not be sequenced using a protein sequencer [8], the a-amino group of the N-terminal residue of mature NDP kinase I appears to be modified. It is possible that such a structural difference between the N-terminal regions of NDP kinases I and II reflects a functional difference between the two enzymes in spinach cells. The homology (complete matching) between the sequences of spinach NDP kinases II and I was calculated to be 58.8% (Fig. 2: amino acid residues of NDP kinase II are numbered from the N-terminal amino acid of the mature NDP kinase II). Striking homology has been observed between the sequence of spinach NDP kinase II, and those of rat NDP kinases a and/3 [5], Dictyostelium discoideum NDP kinase [10], the human Nm23-H1 and Nm23-H2 [6] proteins, and awd protein of Drosophila melanogaster[2]; the homologies with spinach NDP kinase II being 50.3, 51.0, 51.6, 52.3, 49.7 and 53.6%, respectively. It is interesting to note that the sequence homologies between spinach NDP kinase I and the other proteins listed above were 62.8, 63.5, 58.8, 63.5, 62.8 and 66.9%, respectively, which are higher than the sequence homology between spinach NDP kinase I and II. Recently, the X-ray structure of a point mutant of NDP kinase from D. discoideum has been determined [11]. The D. discoideum NDP kinase subunit contained a//3 domain with a four stranded, antiparallel /3-sheet, and the enzyme was composed of six subunits. Sequence homologies between a//3 domain of D. discoideum NDP kinase (amino acid residues 8-138 in Fig. 2) and corresponding domain of spinach NDP kinase I (amino acid residues 4-131 in Fig. 2) or II (amino acid residues 5-135 in Fig. 2) are 57.3% or 64.1%, respectively. This suggests that NDP kinase I and II have simillar a tertiary and quaternary structure to D. discoideum enzyme. The amino acid sequences of possible functional domains in the NDP kinases were also compared. The amino acid residues containing histidine phosphorylation sites [3], Hisll9-Gly-Ser-Asp 122, in spinach NDP kinase II are identical to those in other NDP kinases, including spinach NDP kinase I (Fig. 2). In addition, the sequence of the glycine-rich loop (GlyZ°-Leu-ValGly 23) of spinach NDP kinase II, which is present in

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Fig. 2. Homology between the ~mino acid sequences of spinach NDP kinase 11 and other related proteins. Boxes indicate residues identical to those in the amino acid sequence of spinach NDP kinase II. For comparison, the amino acid residues of NDP kinase II are numbered from the N-terminal amino acid of the purified NDP kinase II, not from the start of the coding region of the cDNA.

various nucleotide-binding proteins [12], is also highly conserved in the NDP kinases. Recently, Kimura et al. reported that rat NDP kinase contains an amino acid sequence highly homologous to the ATP-binding region in cyclic nucleotide-dependent protein kinases [13]. A motif common to protein kinases (Ala-X-Lys), of which the lysine residue is invariant in all protein kinases and is essential for their catalytic activity [12], is also present in spinach NDP kinase I (Ala34-Leu Lys 36) as well as in human Nm23-H2 protein and rat NDP kinase a. Although the function of this sequence in NDP kinase remains to be studied, it probably plays an important role in the catalytic activity of NDP kinase I. On the other hand, a minor difference in amino acid sequence is found in the same region of spinach NDP kinase II. Interestingly, this sequence, Gly38-Leu-Lys4°, is the same as those found in human Nm23-H1 protein and rat NDP kinase /3 (Fig 2). In addition, residues Aspl°4-Phe 1°5 of the spinach NDP kinase I, which also seems to be crucial for catalytic activity [8], is highly conserved in various NDP kinases except for spinach NDP kinase II. In conclusion, spinach NDP kinase II is somewhat different from NDP kinase I in the mode of post-translational modification and in the sequences of possible functional domains.

References

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