Agric. Biol. Chem., 47 (10), 2405~2408, 1983 2405

Rapid Paper B; these two proteins are collectively termed quinolinate synthetase, (2) Protein B converts Synthesis of Quinolinic Acid by aspartic acid to an intermediate capable of the EnzymePreparation of undergoing condensation with DHAP cata- Escherichia coli Which Contains lyzed by Protein A, (3) Proteins A and B are coded by nadA and nadB, respectively, and (4) a Plasmid Carrying the nad the quinolinate synthetase system is subjected Gene for the de novo to feedback inhibition as well as end product Synthesis of NAD repression. An outline of quinolinic acid syn- thesis is shown in Fig. 1. Intermediates of Masaaki Kuwahara, Mizuyo Yonehana, quinolinic acid synthesis, however, have not Tetsuhiro Kimura and Yutaka Ishida yet been isolated in pure forms except for butynedioic acid and its amination product2) Department of Food Science, KagawaUniversity, which are proposed to be intermediates. Our Miki-cho, Kagawa 761-07, Japan experiment aimed to elucidate the inter- Received March 17, 1983 mediary metabolism of the de novo pathway for NADsynthesis using recombinant DNA A plasmid, pNADHl, carrying the nad gene for the de techniques with an E. coli host-vector system. novo synthesis of NADwas constructed with pBR322 and chromosomal DNAof Escherichia coli. Cleavage by re- MATERIALS AND METHODS striction endonucleases showed that the plasmid contained a DNAinsert of 8.9 Kbp at the Hin&lll site ofpBR322. Strains. Escherichia coli C600 (F~, thr~, leu", thi ~, m~, The cell-free extract of a transformant of E. coli C600 r~, LacY) and HB101 carrying plasmid pBR322 were which contained the plasmid formed 5 times more quinol- provided by Prof. A. Kimura, the Research Institute of inic acid from aspartic acid and dihydroxyacetone phos- Food Science, Kyoto University. E. coli AKU 5 was phate than that of C600 did. Enzyme synthesis of quinol- obtained from the stock cultures of the Laboratory of inic acid using partially purified enyzmepreparations Applied Microbiology, Kyoto University. A nicotinic acid showed that the activity of Protein B of quinolinate auxotroph, MY-2, was induced from C600 by nitroso- synthetase increased in the transformant, suggesting that guanidine mutagenesis. Addition of quinolinic acid to the the nadB gene was cloned in the transformant. mediumsupported the growth of this mutant, indicating the lack of the nadA or B gene in this strain. This mutant The de novo pathway for nicotinamide ade- was used as a receptor of recombinant plasmids. MR-4,a nine dinucleotide (NAD) synthesis through revertant strain of a nicotinic acid auxotroph {nadA or B which the precursors aspartic acid and dihy- mutant) or E. coli AKU5, was used as a donor of nad droxyacetone phosphate (DHAP) are led to genes. Unless otherwise stated, C600, MR-4 and transfor- NAD via quinolinic acid was found pre- mants were grown in Davis minimal medium3* supple- mented with leucine (20 mg/liter), threonine (40 mg/liter) dominantly in microorganisms. Accumulated and thiamine (10/ig/liter). MY-2was grown in the same data,1} mostly with the use of Escherichia coli, mediumexcept that nicotinic acid was additionally added showed that (1) quinolinic acid synthesis oc- at a concentration of 0.1 mgper liter. curs on an enzyme complex of Proteins A and Materials. Restriction endonucleases, T4 DNAligase Quinolinate synthetase and X phage DNAwere purchased from Takara Shuzo, Kyoto. Ribonuclease Tj and ribonuclease A were the Protein B Protein A products of Boehringer Mannheim. Bacterial alkaline (nad B) (nad A) r^N-COOH Aspartic * > X 7 > L å *à">NAD acid X / ^COOH phosphatase (Type III-S) was obtained from Sigma. DHAP Ouinolinic acid Contruction of recombinant DNA.Chromosomal DNA of MR-4was prepared by the method of Marmur.4) Fig. 1. Quinolinic Acid Synthesis from Aspartic Acid Plasmid PBR322was isolated from HB101by the method and Dihydroxyacetone Phosphate (DHAP)in the de novo of Clewell and Helinski.5) Hybrid plasmids were con- Pathway for NADBiosynthesis. structed by ligating the endonuclease-digested fragments X, unclarified intermediate. of chromosome and plasmid DNAs6) using T4 DNA 2406 M. Kuwahara et al. ligase.7) The ligated DNAwas used to transform com- 50W x 2 (H+) (1 cm x 18cm) columns successively accord- ing to the method of Chandler and Gholson.10) petent cells8} of MY-2.The transformed cells were selected Step II. The pooled quinolinic acid fractions were on minimal plates containing tetracycline (20 mg/liter) or concentrated with an evaporator and the residue was ampicillin (20 mg/liter). dissolved in 0.2ml of water. The solution was applied on three strips of Whatman3MMand developed with a butanol-acetic acid-water (4 : 1 : 2, v/v) system. Bioassay. Nicotinic acid and the intermediates of the Quinolinic acid on the chromatogram was located under UVlight and extracted with 20ml of water at 37°C for NAD synthetic pathway were analyzed by a micro- lhr. bioassay using MY-2as a test organism. A 0.5ml aliquot Step III. The concentrate of the extract was applied to a of a culture filtrate, obtained by membranefiltration, was Dowex 1 x 2 (formate") column (1 cm x 12cm) and eluted added to 5ml of the medium(nicotinic acid was not added with formic acid with stepwise increments of the formic acid concentration. Fractions containing quinolinic acid in this case) in an 18mmtest tube and 0.15ml ofa cell which was eluted with 2.0n formic acid showed constant suspension of MY-2was inoculated. After 10hr incu- specific radioactivity, indicating that the quinolinic acid separated by this column was radiochemically pure. The bation at 37°C with gentle shaking, growth of the test purity of quinolinic acid was also ascertained by paper organism was measuredusing a spectrophotometer. A chromatography of the concentrate of the quinolinic acid dose-response curve was constructed using nicotinic acid fraction. Recovery of quinolinic acid through these steps was 70 as a reference compoundat concentrations of 0 to 0.1 //g to 75%and the amount of the synthesized quinolinic acid perml. was calculated based ion the radioactivity of quinolinic Preparation of cell-free extract andpartialpurification of acid. quinolinate synthetase. A subculture of each strain was inoculated into 1 liter of the medium in a 5-liter RESULTS AND DISCUSSION Erlenmeyer flask and the culture was carried out at 30°C A transformant strain, C600(pNADHl), with rotary shaking. Cells were harvested after 20hr trophwas obtainedwith theby transformationhybrid plasmid ofDNAthe auxo-con- incubation at the stationary growth phase by centrifu- structed with Hindlll fragments of chromo- gation, washed with 0.05 m potassium phosphate buffer some and pBR322. The plasmid harbored in (pH 8.0) and finally suspended in the same buffer. Cells the transformant was cured with sodium do- were disrupted by sonic oscillation and the supernatant decyl sulfate11} or acridine orange12) with a solution was obtained by centrifugation. Partial purifi- cation of quinolinate synthetase was carried out according to the method of Suzuki et al9) An enzyme solution purified by heat treatment (55°C, 5 min) and ammonium sulfate fractionation (40~60% saturation) was used as a Protein Apreparation. Anenzymesolution obtained by protamine sulfate precipitation and ammoniumsulfate fractionation (0 ~ 40%) was used as Protein B preparation. Quinolinate synthetase assay. The standard mixture for the quinolinate synthetase assay9} contained 1 /zCi of l[U- 14C]-aspartic acid, 0.625 /miol of unlabeled aspartic acid, 2.5/miol of DHAP, 0.1/miol of FAD, 75/miol of Na- bicine (pH 8.0) and enzyme protein (12 to 14mg) in a total volume of 1.25ml. The reaction was carried out at 28°C for 6hr in the dark and the reaction was terminated by addition of 65 fi\ of 2.2 n perchloric acid. After centrifu- '2.9 gation, the supernatant was neutralized by addition of solid KHCO3and centrifuged again. Radiolabeled quinol- inic acid in the deproteinized and neutralized reaction mixture was assayed according to the following pro- Fig. 2. Restriction cleavage Map of pNADHl. cedures. Throughout this column chromatographic sepa- Recombinant plasmid pNADHl was isolated and purified ration, elution of quinolinic acid was monitored by from the transformant C600(pNADHl) and subjected to measuring the absorbance at 268 nmand radioactivity was agarose gel electrophoresis after digestion with single or detected with an Aloka Liquid Scintillation Counter combinations of restriction enzymes. The thick and thin Model LCS 671 using Scintizol EX-H (Kojin, Tokyo) bands represent the pBR322and inserted DNAregions, as a scintillation fluid. respectively. The numerals indicate molecular mass in Kbp Step /. A 0.75ml aliquot of the reaction mixture was of the DNAfragments between each two restriction sites mixed with 0.2ml of 10mM unlabeled quinolinic acid as a of the inserted DNA.Symbolsshow restriction sites for marker compound and then quinolinic acid was frac- EcoRl( - ), Hindlll'(-a), Bam¥L\ ( -«), Pstl (-à") and tionated on Dowex1 x2 (Cl~) (1cmx 12cm) and Dowex Sall (-o). Cloning of nad Gene 2407 Table I. Quinolinic Acid Synthesis 2.05/ig of nicotinic acid-active compound per by Cell-free Extracts of ml of the culture, whereas C600 cells produced E. coli Strains 0.43 fig. Activity of quinolinate synthetase, the os trai. n s , 1M --. .(nmol/1Q.25umlmreaocthinonmiicxtaurcei)d synthesized only known enzyme in the de novo pathway, was assayed. As shown in Table I, the cell-free MR - 4 (R ev e rt an t) 0. 3 extract of C600(pNADHl)produced 5 times C 6 0 0 0 . 3 morequinolinic acid than that produced by M Y -2 (m c~ ) 0.05 C600 and MR-4. Production by MY-2 was C 6 0 0 ( P N A D H l ) ( T r a n s f o r m a n t ) extremely low. This result indicated the am- plification of the nadA or B gene in the transformant. Table II. Quinolinic Acid Synthesis To determine the enzyme whose activity was by Proteins A and B Partially increased by transformation, quinolinate syn- Purified f rom C600(PNAD H l) thesis was examined with the partially purified and MY-2 (nic ) enzyme prepared from C600(pNADHl)and Combination of Quinolinic acid synthesized MY-2. The results are presented in Table II. enzymes (nmol/1.25 ml reaction mixture) Whenfractions of Proteins Aand B of the transformant were mixed (TA+TB) and in- T A + T B 6 .0 cubated with aspartic acid and DHAP, M A + M B 1.2 M A + T B 4 .9 6.0nmol of quinolinic acid was formed. T A + M B 1.2 Reactions with a single enzyme, Protein A T A 1.0 (TA) or Protein B (TB), gave 1.0 or 0.2nmol, M A 1.1 respectively. Therefore, the net formation of T B 0.2 M B 0.3 quinolinic acid by TA+TB reaction was 4.8mmol. Onthe other hand, in the reaction TA, Protein A from C600(pNADHl) (16mg); TB, with proteins A plus B of MY-2 (MA+MB), Protein B from C600(pNADHl) (15mg); MA, Protein A in which either one of the proteins might not from MY-2 (16mg); MB, Protein B from MY-2 (15mg). possess the activity, a low level of quinolinic acid was formed, combination of Protein A concomitant loss of tetracycline resistance and from MY-2and Protein B from the transfor- the regaining of nicotinic acid auxotrophy. mant (MA+TB) produced quinolinic acid, The isolated plasmid (pNADHl) from the whereas the reverse combination (TA+MB) nicotinic auxotroph transformant was digested did not. This result suggested that MY-2 lack- with EcoRI, Hindlll, BamHI, Sail and Pstl ed Protein B and the activity of the protein was compensated for by nadB coded on the agaroseand the resultinggel electrophoresis13)fragments were(0.7%analyzedagarose by recombinant plasmid pNADHl. Auxotrophy gel) with i7/ftdIII-digests of' X phage DNA as a is often found to be suppressed by a mutation molecular weight standard. The pNADHlcon- in a second gene. However, suppression of tained an insert (approximately 8.9Kbp) of nicotinic acid auxotrophy in E. coli has not DNA at the Hin&lll site of pBR322. The been recognized so far. The increase in the inserted DNA contained a single site for activity of quinolinate synthetase in the trans- EcoRl and BamHIand two SalI sites, the formant is unlikely to be caused by a sup- proposed restriction map of pNaDHlis shown pressor gene, but rather the nadBgene. in Fig. 2. Experiments on the de novo pathway have Filtrates of the 24-hr cultures of C600 and been performed mostly by using E. coli mu- C600(pNADHl) were subjected to the bio- tants defective in this pathway. Results ob- assay. The assay showed that 1 mg (as dried tained in this study suggest that nadB for matter) of the transformant cells excreted quinolinate synthetase was cloned in E. coli 2408 M. Kuwahara et al. using vector plasmid pBR322. Wethink that D. Davis, L. M. Henderson and D. Powel, J. Biol. this study is the first step in the cloning of the Chem., 189, 543 (1951). whole NAD synthetic gene system, which J. Marmur, /. Mol Biol, 3, 208 (1961). D. B. Clewell and D. R. Helinski, Biochemistry, 62, could lead to improvementin the production 1159 (1969). of NAD. J. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A., 69, 339 (1972). Acknowledgments. Weare grateful to Professor A. T. Maniatis, E. F. Fritsch and J. Sambrock, Kimura and Dr. K. Murata, the Research Institute for "Molecular Cloning," Cold Spring Harbor Labo- Food Science, Kyoto University, for their useful advice ratory, Cold Spring Harbor, NewYork, 1982, p. and discussions as well as for providing bacterial strains. 391. 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