Adenylate Kinase Complements Nucleoside Diphosphate Kinase

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5720-5725, June 1996 Biochemistry Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism QING LU AND MASAYORI INOUYE* Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854 Communicated by Arthur Kornberg, Stanford University Medical Center, Stanford, CA, February 21, 1996 (received for review January 29, 1996) ABSTRACT Nucleoside diphosphate (NDP) kinase is a (17), which suggests the existence of an interesting unidenti- ubiquitous nonspecific enzyme that evidently is designed to fied enzyme(s) that complements the NDP kinase activity for catalyze in vivo ATP-dependent synthesis of ribo- and deoxyri- (d)NTPs synthesis in the cell. bonucleoside triphosphates from the corresponding diphos- In this study, we attempted to identify the enzyme that phates. Because Escherichia coli contains only one copy ofndk, complements the NDP kinase function in the ndk-disrupted the structural gene for this enzyme, we were surprised to find strains ofE. coli. NDP kinase activity of the ndk cell extract was that ndk disruption yields bacteria that are still viable. These 10-15% of that of the wild-type cell extract (17). Because mutant cells contain a protein with a small amount NDP there is only one ndk gene on the E. coli chromosome (18), the kinase activity. The protein responsible for this activity was residual NDP kinase activity may be attributed to a novel NDP purified and identified as adenylate kinase. This enzyme, also kinase. There are two possible enzymes that may complement called myokinase, catalyzes the reversible ATP-dependent NDP kinase activity in E. coli: pyruvate kinases (PykA and synthesis ofADP from AMP. We found that this enzyme from PykF; ref. 19) and succinyl-CoA synthetase (Scs), that may E. coli as well as from higher eukaryotes has a broad substrate form a complex with NDP kinase (20). However, we have specificity displaying dual enzymatic functions. Among the constructed an ndk pykApykF triple mutant as well as an ndk nucleoside monophosphate kinases tested, only adenylate Scs double mutant and demonstrated that they still grew kinase was found to have NDP kinase activity. To our knowl- normally without apparent morphological changes (17). Re- edge, this is the first report of NDP kinase activity associated sidual NDP kinase activities in these triple and double mutants with adenylate kinase. were comparable to the activity in the single ndk-disrupted cells, indicating that neither pyruvate kinases nor Scs is the Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme complementing NDP kinase in the ndk cells. We now dem- to catalyze the ATP-dependent synthesis of ribo- and deoxyri- onstrate that this complementing enzyme is adenylate kinase, bonucleoside triphosphates from the corresponding diphos- a monophosphate kinase known to be essential for the gen- phates via a phosphoenzyme intermediate. It has a broad eration of ADP from AMP and normal cell growth. specificity for phosphoryl donors and acceptors to maintain the balanced levels of nucleoside triphosphates in the cell (1, MATERIALS AND METHODS 2). The structures of NDP kinases are highly conserved from Escherichia coli to human (43% identity), and they are believed Bacterial Strains and Plasmids. E. coli strain QL7623, an to be a housekeeping enzyme essential for DNA and RNA ndk-disrupted strain derived from strain JC7623 (17) and strain synthesis (2-4). In addition, NDP kinases have been shown to BL21(DE3) were used. The latter strain was obtained from New have additional regulatory functions for growth and develop- England Biolabs. Cells were grown in Luria-Bertani (LB) me- mental control (5-9), signal transduction (10, 11), and tumor dium (for strain JC7623 and QL7623) or in M9 minimal medium metastasis suppression (12, 13). Human and mouse NDP [for strain BL21(DE3)] at 37°C. When necessary, chloramphen- kinases/Nm23 have been considered to play an important role icol (25 tLg/ml) and ampicillin (50 ,pg/ml) were added. in tumor metastasis suppression. High metastatic potential of Plasmid pKX110 and plasmid pKX7 were obtained from K. low nm23-expressing murine melanoma and human breast Yamanaka (21). Plasmid pKX110 carries the wild-type smbA carcinoma cell line was inhibited with transfection of nm23 (umk) gene and plasmid pKX7 carries the wild-type mssA (cmk) cDNA (4, 14). A human DNA-binding protein, PuF, identified gene. as NDP kinase Nm23-H2, was shown to bind to the promoter Detection of the Residual NDP Kinase Activity in the ndk region of c-myc in vitro and to activate the c-myc transcription, Cells. Cell extracts obtained by sonication from a 2-ml over- which suggested its regulatory role in oncogene expression night culture of strain QL7623 were incubated at 55°C for 5 (15). The cytoplasmic membrane of Dictyostelium discoideum min in the sample loading buffer (80 mM Tris-HCl, pH 6.8/1% possesses a cAMP receptor-stimulated NDP kinase activity SDS/10% glycerol/4% 2-mercaptoethanol/0.005% bromo- that may contribute to mediation of hormone action for the phenol blue). SDS/PAGE was carried out with use of a 15% activation of G proteins (10). A null mutation of the awd gene, polyacrylamide gel at 4°C with constant current of 7 mA. After a Drosophila homologue of NDP kinase, results in abnormal- electrophoresis the gels were sliced into 3 mm (see Fig. 1A) or ities in the larvae development (6). A point mutation of Awd 4 mm (see Fig. 1B) segments. Each segment was then sepa- leads to a lethal phenotype when it is put together with a rately extracted with 200 ,tl renaturation buffer {100 mM mutation in the prune gene (16). Studies of NDP kinase from Hepes, pH 8.0/2% glycerol/0.1% 3-[(3-cholamidopropyl)dim- a have ethylammonio]-l-propanesulfonate] (CHAPS)/1 mM dithio- Myxococccus xanthus, gram-negative bacterium, sug- threitol (DTT)} for 48 hr at 4°C. Ten microliters of the extract gested that the enzyme may be essential for cell growth (5). was used for the NDP kinase as described 1 Surprisingly, however, it was found that the ndk gene, the only assay (22), using structural gene for this enzyme in E. coli, could be disrupted mM dCDP as substrate and 5 ,Ci [y-32P]ATP (6000 Ci/mmol; without affecting cell viability except for a mutator phenotype Abbreviations: NDP, nucleoside diphosphate; Scs, succinyl-CoA- synthetase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l- The publication costs of this article were defrayed in part by page charge propanesulfonate. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. e-mail: inouye@rwja. accordance with 18 U.S.C. §1734 solely to indicate this fact. umdnj.edu. 5720 Downloaded by guest on October 2, 2021 Biochemistry: Lu and Inouye Proc. Natl. Acad. Sci. USA 93 (1996) 5721 1 Ci = 37 GBq) as phosphate donor in a 20-,.l reaction mixture BamHI to generate plasmid pETadk. The entire region of adk containing 50 mM Hepes (pH 8.0), 50 mM NaCI, 10 mM insert in pETadk was sequenced to confirm that no errors were MgCl2, and 1 mM DTT. After 2 hr incubation, 1 ,l of the generated by the PCR process. The BL21(DE3) ndk::cmr strain reaction mixture was loaded on a polyethyleneimine-cellulose was transformed with pET-adk. The maximal induction of plate. Synthesis of [y-32P]dCTP was analyzed by ascending adenylate kinase was obtained in M9 minimal medium at 37°C chromatography with 0.75 M KH2PO4 (pH 3.5) and quanti- for 4 hr in the presence of 2 mM isopropyl P-D-thiogalactoside. tated by a Molecular Imager GS-250 (Bio-Rad). Adenylate kinase was produced at a level of -60% total Purification ofthe Complementary NDP Kinase (Sdk) from cellular protein. The enzyme was then purified basically by the ndk Cells. Whole cell extracts were prepared from 6 liters of same methods as described (24) with some modifications. the ndk-disrupted strain QL7623 (ndk::cmr) grown in LB Briefly, a cell extract from a 500-ml culture was subjected to medium overnight. Cells were harvested and washed once with ammonium sulfate precipitation. The 30-65% precipitate buffer A (50 mM Tris-HCl, pH 8.0/50 mM NaCl/1 mM DTT) fraction was resuspended in 2 ml buffer B (50 mM Tris-HCl, and lysed in 40 ml of buffer A with a French press (at 12000 pH 8.0/1 mM DTT/10% glycerol) and loaded to a 200-ml psi, three times). Cell debris and the membrane fraction were Sephacryl S200 column equilibrated with the same buffer with removed by centrifugation at 45,000 rpm for 90 min in a a flow rate 0.4 ml/min. Fractions containing adenylate kinase Beckman Ti50 rotor. To the supernatant fraction (NH4)2S04 were pooled and loaded directly onto a 30-ml Q-Sepharose was added to 65% saturation. The resulted precipitates were column equilibrated with buffer B and eluted with a 0-0.3 M collected by centrifugation and dissolved in 10 ml of buffer A. KCI gradient. Adenylate kinasewas eluted at a salt range between The solution was extensively dialyzed with buffer A, then 50 and 70 mM. Active fractions were pooled and dialyzed briefly loaded onto a Q-sepharose column (Pharmacia), and equili- against buffer B. The dialyzed solution was then subjected to an brated in buffer A. The column was then washed with buffer FPLC polyethyleneimine anion exchange-1000 column with a A containing 100 mM KCI. Fractions eluted with 200 mM KCl gradient of 0-0.3 M KC1. Adenylate kinase eluting from this in buffer A were found to contain the major NDP kinase column was -90% pure, as determined with SDS/PAGE fol- activity.

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