- Email: [email protected]
Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coli
FEMS Mw'oblology Letters 66 (1990) 231-234 Pubhshed by Elsevter
23)
FEMSLE 03822
Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coil G e r d Katzenmeier, Cornelia S c h m i d a n d Adelbert Bacher Lehrstuhl fur Orgunasche Cherme und Brochemw, Techmsche Umversttot Munchen, Garchm~ F R G
Recetved 13 June 1989 Rev,slon recetved 16 August 1989 Accepted 30 August 1989 Key words: Eschench:a coh; GTP-cyclohydrolase 1, Plasmid; Cloning
1. S U M M A R Y The putative gene coding for G T P cyclohydro10se I of Escherlchla cob was isolated from a ?~gtll expression vector library by using antibodies as ~ probe and has been subcloned on a 3.8 kb B a m H I fragment m the plasmid vector pUC13. E. coh cells carrying the recombinant plasrmd destgnated pCYH express 100-fold increased levels of the enzyme. The protein formed under the control of the plasmid appears electrophorettcally and tmmunochemlcally tdentical wtth the wild type enzyme.
2. I N T R O D U C T I O N G T P eyclohydrolase I catalyzes the formatmn of dihydroneopterln tnphosphate from G T P by a Correspondence to G. Katzennuner. Lehrsluhl fur Orgamsche
Chemte und Bio~.netrtte. Techmsche Umversa~it Munchen. Ltchlenbetstr 4.13-8046 Garchm&F R G Abbtevtauons HPLC. tngh performance hqmd chromatography. ATCC. American Type Culture Collecuon. DSM. Deulsche Sammlungyon Mdcroorgamsmen
complex series of reacUons [1]. The reaction represents the first committed step of tetrahydrofolate biosynthesis in many rmeroorganisms [2]. The enz~,me from Escherwhla coil Is a 210 k D a protein, and the quaternary structure appears to be a tetramet of dimers [3]. The primary structure of the protem is unknown. Although vertebrates are unable to form tetrahydrofolate de novo, they possess G T P cyclohydrolase I acuvity catalyzing the first committed step m the biosynthesis of tetrahydroblopterin wl~ch serves as cofactor for the hydroxylation of aromattc amino acids and is thus involved in the biosynthesis of catecholammes [4]. Moreover, tetrahydrubtopterm appears to be involved in Tlymphocyte activaUon [5]. G T P eyclohydrolase I has been obtained in highly purified form from human liver [6]. The enzyme was very unstable, and Mile is known about its structure. It has been reported that several monoclonal antibodies directed against the human enzyme crossreacted with the enzyme from E. colt [6]. "t~s paper describes the cloning of the putative gene coding for G T P eyclohydrolase I of E. col:. The expression of the gene m E. cob allows the tsolation of large amounts of pure protem for
0378-1097/89/$03 50 © 1989 Federauon of European Mlcrobsolo~cal Soclettes
232 detailed structural and mechanistic studies To the best of our knowledge, no other gene coding for GTP cyelohydrolase I has been cloned batherto.
M NaC1 (0.5 ml). The suspension was stirred at room temperature for 2 h and washed with a solution containing 0.1 M sedmm acetate pH 4.0 and 0.5 M NaCI followed by 0.1 M "Ins hydrochloride pH 8.0 containing 0.5 M NaCI.
3. MATERIALS 3.1. Bacterial strains The following stratus been used: strain Y1090 for Xgtll, strain JM83 (Pharmaela), and strata chromosomal DNA for struction.
of Eschertchta cob have ATCC 37197 [7] as host [8] as host for pUC13 LE392 [9] as source of the ~.gtll library con-
3.2. Chemwals Pteridines used were purchased from Dr. B. Schircks, Jona, Swazedand. Other chemicals were of the tughest purity available. 3.3. Enzymes GTP cyelohydrolase I was purified from cells of E. cob by the protocol of Ylm and Brown [3[. Restriction nucleases and other enzymes were purchased from Boehrmger, Pharmacia, and Glbco. 3.4. Enzyme assay The assay mixture for the deternunation of GTP cyclohydrolase I actwity contained 0.1 M Tns hydrochlonde pH 8.5, 0.1 M KCI, 2.5 mM EDTA, 0.4 mM GTP, and protein in a total volume of 250 ~1. Assays were incubated at 37°C for 30 rain and terminated by the addition of 1 M HCI (40 /d) contalmng 1% 12 and 2% KI. The solutmn was neutralized, alkaline phosphatase (3 U) was added, and the nuxture was incubated for 15 rain at 37°C. Neop~a'm was analyzed by reversed phase HPLC (Nucleosd 10 RPI8 column, 4 × 250 nun; eluent, 7'$ methanol with 30 mM formic acid). Elutioa was monitored fluorometri-
caily, 3.5. Preparatmn of tmmoblhzed GTP cyclohydrolase 1 GTP cyclohydrolase I (1 rag) was added to BrCN-activated Sepharose 4B suspended m 0.1 M sodium bicarbonate buffcx pH 8.5 containing 0.5
3.6. AnubodJes Antiserum was prepared by repeated imnlumzaUon of a rabbR with 0.25 mg ahquots of GTP cyclohydrolase I. For the initial unmunization, the antigen was appfied in Freund's complete adjuvant. The serum (3 mi) was appfied to a column of immobilized enzyme (0.5 ml), and the column was washed with 25 mi of 10 mM phosphate pH 7.4 contaimng 0.63 M NaCI and 0.05~ Tween. Subsequently, the column was washed with 20 mi of 30 mM NaCI and developed with 0.1 M glycine hydrochlonde pH 2.5. Fractions were monitored by immunoblotting. Fractions were pooled and concentrated by ultrafiltratlon. 3. 7. Electrophoresis and lmmunoblottmg SDS polyacrylanude gel electrophoresis (SDSPAGE) was carried out according to Laemmll [10]. Proteins were separated on 7.5-20~ gradient gels and visualized by staining with Coomassie Brilliant Blue. Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes according to [11] and probed wRh affinity purified antthodies [12]. 3.8. DNA methods Isolation of plasmtd and ~DNA, restriction endanuclease digestion, ligatlon and transformatmn of E. coh strains were performed as described by Maniatis [13]. The ~.gtll library was packaged by using a packaging kit from Atlanta, Heidelberg. 3.9. Screemng of phage hbrarws The ~gtll library was plated on E. ceh Y1090 and screened with immunopunfied rabbit antiserum raised against E. coh GTP ¢yclohydrolase I as described by Mierendorf et al. [12]. Immune complexes were detected w~th alkaline phosphatase labeled goat antirabbit IgG (Atlanta Inc.). Screening was repeated until all plaques were posttive.
233 3.10. Constructton of recombmam plasmlds The recombinant D N A of Agtll immunopostttve clones were isolated and digested vath BamHI under standard con&tions. Samples were mixed vath BamHl-,hgested and phosphatase treated p U C I 3 plasmid and hgated vath T4 DNA-figase.
1
2
3
4
---
|
4. RESULTS G T P cyclohydrolase 1 was purified t o electrophoretic homogeneity from E. cob strain DSM 613 and was used to tmmuarze a rabbit. The anttserum was purified by lmmanochromatography on immobihzed G T P cyclohydrolase 1. The Western blot of E. coh cell extract showed a single band corresponding to M r = 24000 (Ftg 1). A gene bank was generated by digesting E cola LE392 D N A wtth EcoRl and msertmg the fragments into the EcoRI site of phage Agtll accordm g m the procedure of Huynh ¢t al. [14]. Agar plates with an average of 1000 phage plaques were prepared and screened by immunoblottmg usm 8 affinity-purified polyclonal antibodies, A total of 6 positive clones was obtained. Phage D N A of 4
1
2
3
4
66-
45--
~
36--
3.11. Isolauon of GTP cyclohydrolase I from E colt JM83 carrying the plasmtd p C Y H Frozen cells (40 g) were thawed m 170 ml of a solution containing 0.2 M ' I n s hydrochlonde pH 8.0, 2.5 m M EDTA, 40 mg of lysozyme, and 4 mg of DNase. l-he suspension was incubated for 1.5 h at 3 7 ° C and centrifuged. To the supernatant, ammonium sulfate was added to a final saturation of 30~. The precipitate was removed by cenmfugation and dtscarded. The supematant was brought to 50~ saturation by the addmon of sohd amm o m u m sulfate, and the precipttate was dtssolved m 50 m M ' I n s hydrochlonde pH 8.0 containing 0.3 M KCI. The solutton was dialyzed against the same buffer and was subsequently placed on a c o l u m of Sephadex G-75 (2.5 × 90 cm) The colunto was developed wtth 50 mM T n s hydrochloride pH 8.0 containing 0.3 M KC1. The fra,,tlon from 189 to 240 ml was apphed to a column of G T P Sepharose which was developed wtth 100 m[ of 10 mM phosphate pH 7.0 contammg 2.5 m M E D T A and 0.5 mM G T P [3].
5
20-t4--
~
A
B
Fig. I SDS polyacrylarmde gel electrophoresJsand co~espond. mg Western tmmunoblot A, Coomasst¢ blue-stmncd electro*
pherogram, I, molecularwe=ght markers(70 pg), 2 and 5, pure GTP-cyclohydrolase 1 (4 pg), 3, cell lysate of E cob JM83 (parent strain. 45 #tg), 4, cell lysate of E co/: JMg3 pCYH (55 ~tg) Molecularweight markers (kDal are indicated on the left B, immunoblot, t and 4, purified GTP cyclohydrolase l. 2. cell lysate of E colt JM83 pCYH. 3, cell lysate of E colt JM83
different clones was prepared and analyzed by agarose electrophorests after digestton with EcoRL Each of the clones analyzed contained an insert of approximately 9.0 kb and tt appeared that all isolates were identical. The recombinant D N A of one Agtll clone was purified and digested with BamHl, and the resultmg fragments were ligated into the BamHl site of the plasnud vector pUC13. E colt JM83 was transformed and the ampictllin reststant colonies were again screened by immunoblottmg [15]. Two posture colonies were isolated. Crude cell extracts were prepared and analyzed for G T P cyclohydrolase 1 activity which was found about 100-fold mcreased as compared to the parent strain, JM83. Cell extracts were subjected to SDS electrophoresls followed by Western blotting (Fig. 1) Both clones showed strong tmmunoreactive bands corresponding to M r = 24000 which appeared identical with the wild-type enzyme. The plasmids obtained from the clones analyzed showed the presence of a 3.8 kb insert upon resmctton with BamHI. Plasmtd pCYH was used m subsequent work. A preliminary restrmtion map of the insert is shown in Ftg. 2. The msert contains no cutoog rotes for the following enzymes: Aval. Sacl, BanII, XbaI.
234 BH
I I t
lkb
P
B
I
I
i
Fig 2 Restncuonmap of the 38kb BamH[ insert containing the cloned GTP ¢yclohydrolase 1 gene Plasrmd pCYH was obtained by subclomngof one of the BamHI fragments from Agtll clone m pUCI3 Enzyme sites are indicated by a one letterdesignation(B, BamHI, P, /~stl,H, HmdlII) From 40 g of cells of E. colt JM83 pCYH, we could isolate 100 mg of pure GTP cyclohydrolas¢ I usmg the protocol of Yim and Brown with the modihcattong described under MATERIALS AND METHODS. The protein appeared identical with the wild type enzyme as judged by SDS gel electrophoresis. By comparison, 8 mg of enzyme were obtained from 500 g of E, cob wild type cells.
5. DISCUSSION The presence of a 3.8 kb BaraHl fragment of E. colt in a pUCI3 plasrmd leads to an apprmamately 100 fold increased level of GTP cyclohydrolase I activity in E, colt. The enzyme isolated from the plaslmd-carrymg strain has a subunit molecular weight of 24000 and appears identical with the wild type protein. Tins suggests that the 3.8 kb BamHI fragment harbors the entire structural gene for GTP ¢yclohydrolase I including the promoter and the termination signal. We cannot formally rule out the possibihty that the 3.8 kb fragment codes for an activator protein conducing to a dramatic increase In the expression of the chromosomal GTP cyclohydrolase I gene, but the hypothesis appears rather unlikely, since there is at present no evidence for regulauon of any type of GTP cyclohydrolase I in E. colt. The preparation of GTP cyclohydrolase I t s greatly facilitated by the increased enzyme levels contained in the plasmid harboring strains. There is no evidence for a negative effect of the increased enzyme level on the viabihty of the organism. More detailed studies of the structure and function of the enzyme should become pnssi-
ble by the improved availability of the protein. Moreover, the enzyme is a useful reagent for the preparation of dihydroneopt.erin 3'-tnphosphate for which there is increased demand as a consequence of the novel interest in the biosynthesis and ragulauon of unconjugated ptertns in mammals.
ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgememschaft and the Fond der Chemlschen lndustne. We thank Drs. F. GiStz and A. BiSck for helpful discussions.
REFERENCES [ll Brown, (3 M and Wdhamson, J M (1982)Adv Enzymol 53, 345-35L [2} B]au, N. and Nlederwleser, A (1985) J Chll Chem. CHIn.
BlOchem 23,169-176 [3] Ylm, J J and Brown, G M (1976) J Btol Chem 251, 5087-5094 [4] Ntcbol,C A, Smith, G K and l)ud~ D S (1985lAnnu Rev Blochern 54, 729-764, 151 Z~egler,I (1987)Naturwssenschaften 74, 563-572
[6] Schoedon, G.S, Redwelk, U and Curtius, H C. (1989) Eur J Bsochem 1"/8,627-634 [7i Young,R A and Davis,R W (1983)Scmnce222,778-782 [8] Yamsch-Perron, C. Vlelra,J. and Messing,J (1988)Gene 33,103-119. [9] Murray, N.E. Brammer, WJ and Murray, K (197"/) Melee Gen Genet 150,53-61 [10] Laemmh,U.K, (19'/0) Nature 227, 680-685 [11] Towbm, H., Staehhn, T and Gordon, J (1979)Proc Nat #.cad Sol USA 76, 4350-4354 [12] Mte~ndorL Re,, Percy, C and Young, R,A (1987) M¢lhOdsEnzymol 152,458-469 [131 Mamaus, T. Fntsch, E,F, and Sambn~ok, I (1982) Molecular clomng" A laboratory manual Cold Spnng Harbor Laboratory, New York [14] Huynh, TV, Young, R.A and Davis, R.W (1985) m DNA Cloning,'4ol 1 (Glover.D M, ed ), pp 49-78, IRL Press Lid,, Oxford 115l Hdfman, D,M, and Hughes, SH, (1987) Methods Enzymol 152,451-457
23)
FEMSLE 03822
Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coil G e r d Katzenmeier, Cornelia S c h m i d a n d Adelbert Bacher Lehrstuhl fur Orgunasche Cherme und Brochemw, Techmsche Umversttot Munchen, Garchm~ F R G
Recetved 13 June 1989 Rev,slon recetved 16 August 1989 Accepted 30 August 1989 Key words: Eschench:a coh; GTP-cyclohydrolase 1, Plasmid; Cloning
1. S U M M A R Y The putative gene coding for G T P cyclohydro10se I of Escherlchla cob was isolated from a ?~gtll expression vector library by using antibodies as ~ probe and has been subcloned on a 3.8 kb B a m H I fragment m the plasmid vector pUC13. E. coh cells carrying the recombinant plasrmd destgnated pCYH express 100-fold increased levels of the enzyme. The protein formed under the control of the plasmid appears electrophorettcally and tmmunochemlcally tdentical wtth the wild type enzyme.
2. I N T R O D U C T I O N G T P eyclohydrolase I catalyzes the formatmn of dihydroneopterln tnphosphate from G T P by a Correspondence to G. Katzennuner. Lehrsluhl fur Orgamsche
Chemte und Bio~.netrtte. Techmsche Umversa~it Munchen. Ltchlenbetstr 4.13-8046 Garchm&F R G Abbtevtauons HPLC. tngh performance hqmd chromatography. ATCC. American Type Culture Collecuon. DSM. Deulsche Sammlungyon Mdcroorgamsmen
complex series of reacUons [1]. The reaction represents the first committed step of tetrahydrofolate biosynthesis in many rmeroorganisms [2]. The enz~,me from Escherwhla coil Is a 210 k D a protein, and the quaternary structure appears to be a tetramet of dimers [3]. The primary structure of the protem is unknown. Although vertebrates are unable to form tetrahydrofolate de novo, they possess G T P cyclohydrolase I acuvity catalyzing the first committed step m the biosynthesis of tetrahydroblopterin wl~ch serves as cofactor for the hydroxylation of aromattc amino acids and is thus involved in the biosynthesis of catecholammes [4]. Moreover, tetrahydrubtopterm appears to be involved in Tlymphocyte activaUon [5]. G T P eyclohydrolase I has been obtained in highly purified form from human liver [6]. The enzyme was very unstable, and Mile is known about its structure. It has been reported that several monoclonal antibodies directed against the human enzyme crossreacted with the enzyme from E. colt [6]. "t~s paper describes the cloning of the putative gene coding for G T P eyclohydrolase I of E. col:. The expression of the gene m E. cob allows the tsolation of large amounts of pure protem for
0378-1097/89/$03 50 © 1989 Federauon of European Mlcrobsolo~cal Soclettes
232 detailed structural and mechanistic studies To the best of our knowledge, no other gene coding for GTP cyelohydrolase I has been cloned batherto.
M NaC1 (0.5 ml). The suspension was stirred at room temperature for 2 h and washed with a solution containing 0.1 M sedmm acetate pH 4.0 and 0.5 M NaCI followed by 0.1 M "Ins hydrochloride pH 8.0 containing 0.5 M NaCI.
3. MATERIALS 3.1. Bacterial strains The following stratus been used: strain Y1090 for Xgtll, strain JM83 (Pharmaela), and strata chromosomal DNA for struction.
of Eschertchta cob have ATCC 37197 [7] as host [8] as host for pUC13 LE392 [9] as source of the ~.gtll library con-
3.2. Chemwals Pteridines used were purchased from Dr. B. Schircks, Jona, Swazedand. Other chemicals were of the tughest purity available. 3.3. Enzymes GTP cyelohydrolase I was purified from cells of E. cob by the protocol of Ylm and Brown [3[. Restriction nucleases and other enzymes were purchased from Boehrmger, Pharmacia, and Glbco. 3.4. Enzyme assay The assay mixture for the deternunation of GTP cyclohydrolase I actwity contained 0.1 M Tns hydrochlonde pH 8.5, 0.1 M KCI, 2.5 mM EDTA, 0.4 mM GTP, and protein in a total volume of 250 ~1. Assays were incubated at 37°C for 30 rain and terminated by the addition of 1 M HCI (40 /d) contalmng 1% 12 and 2% KI. The solutmn was neutralized, alkaline phosphatase (3 U) was added, and the nuxture was incubated for 15 rain at 37°C. Neop~a'm was analyzed by reversed phase HPLC (Nucleosd 10 RPI8 column, 4 × 250 nun; eluent, 7'$ methanol with 30 mM formic acid). Elutioa was monitored fluorometri-
caily, 3.5. Preparatmn of tmmoblhzed GTP cyclohydrolase 1 GTP cyclohydrolase I (1 rag) was added to BrCN-activated Sepharose 4B suspended m 0.1 M sodium bicarbonate buffcx pH 8.5 containing 0.5
3.6. AnubodJes Antiserum was prepared by repeated imnlumzaUon of a rabbR with 0.25 mg ahquots of GTP cyclohydrolase I. For the initial unmunization, the antigen was appfied in Freund's complete adjuvant. The serum (3 mi) was appfied to a column of immobilized enzyme (0.5 ml), and the column was washed with 25 mi of 10 mM phosphate pH 7.4 contaimng 0.63 M NaCI and 0.05~ Tween. Subsequently, the column was washed with 20 mi of 30 mM NaCI and developed with 0.1 M glycine hydrochlonde pH 2.5. Fractions were monitored by immunoblotting. Fractions were pooled and concentrated by ultrafiltratlon. 3. 7. Electrophoresis and lmmunoblottmg SDS polyacrylanude gel electrophoresis (SDSPAGE) was carried out according to Laemmll [10]. Proteins were separated on 7.5-20~ gradient gels and visualized by staining with Coomassie Brilliant Blue. Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes according to [11] and probed wRh affinity purified antthodies [12]. 3.8. DNA methods Isolation of plasmtd and ~DNA, restriction endanuclease digestion, ligatlon and transformatmn of E. coh strains were performed as described by Maniatis [13]. The ~.gtll library was packaged by using a packaging kit from Atlanta, Heidelberg. 3.9. Screemng of phage hbrarws The ~gtll library was plated on E. ceh Y1090 and screened with immunopunfied rabbit antiserum raised against E. coh GTP ¢yclohydrolase I as described by Mierendorf et al. [12]. Immune complexes were detected w~th alkaline phosphatase labeled goat antirabbit IgG (Atlanta Inc.). Screening was repeated until all plaques were posttive.
233 3.10. Constructton of recombmam plasmlds The recombinant D N A of Agtll immunopostttve clones were isolated and digested vath BamHI under standard con&tions. Samples were mixed vath BamHl-,hgested and phosphatase treated p U C I 3 plasmid and hgated vath T4 DNA-figase.
1
2
3
4
---
|
4. RESULTS G T P cyclohydrolase 1 was purified t o electrophoretic homogeneity from E. cob strain DSM 613 and was used to tmmuarze a rabbit. The anttserum was purified by lmmanochromatography on immobihzed G T P cyclohydrolase 1. The Western blot of E. coh cell extract showed a single band corresponding to M r = 24000 (Ftg 1). A gene bank was generated by digesting E cola LE392 D N A wtth EcoRl and msertmg the fragments into the EcoRI site of phage Agtll accordm g m the procedure of Huynh ¢t al. [14]. Agar plates with an average of 1000 phage plaques were prepared and screened by immunoblottmg usm 8 affinity-purified polyclonal antibodies, A total of 6 positive clones was obtained. Phage D N A of 4
1
2
3
4
66-
45--
~
36--
3.11. Isolauon of GTP cyclohydrolase I from E colt JM83 carrying the plasmtd p C Y H Frozen cells (40 g) were thawed m 170 ml of a solution containing 0.2 M ' I n s hydrochlonde pH 8.0, 2.5 m M EDTA, 40 mg of lysozyme, and 4 mg of DNase. l-he suspension was incubated for 1.5 h at 3 7 ° C and centrifuged. To the supernatant, ammonium sulfate was added to a final saturation of 30~. The precipitate was removed by cenmfugation and dtscarded. The supematant was brought to 50~ saturation by the addmon of sohd amm o m u m sulfate, and the precipttate was dtssolved m 50 m M ' I n s hydrochlonde pH 8.0 containing 0.3 M KCI. The solutton was dialyzed against the same buffer and was subsequently placed on a c o l u m of Sephadex G-75 (2.5 × 90 cm) The colunto was developed wtth 50 mM T n s hydrochloride pH 8.0 containing 0.3 M KC1. The fra,,tlon from 189 to 240 ml was apphed to a column of G T P Sepharose which was developed wtth 100 m[ of 10 mM phosphate pH 7.0 contammg 2.5 m M E D T A and 0.5 mM G T P [3].
5
20-t4--
~
A
B
Fig. I SDS polyacrylarmde gel electrophoresJsand co~espond. mg Western tmmunoblot A, Coomasst¢ blue-stmncd electro*
pherogram, I, molecularwe=ght markers(70 pg), 2 and 5, pure GTP-cyclohydrolase 1 (4 pg), 3, cell lysate of E cob JM83 (parent strain. 45 #tg), 4, cell lysate of E co/: JMg3 pCYH (55 ~tg) Molecularweight markers (kDal are indicated on the left B, immunoblot, t and 4, purified GTP cyclohydrolase l. 2. cell lysate of E colt JM83 pCYH. 3, cell lysate of E colt JM83
different clones was prepared and analyzed by agarose electrophorests after digestton with EcoRL Each of the clones analyzed contained an insert of approximately 9.0 kb and tt appeared that all isolates were identical. The recombinant D N A of one Agtll clone was purified and digested with BamHl, and the resultmg fragments were ligated into the BamHl site of the plasnud vector pUC13. E colt JM83 was transformed and the ampictllin reststant colonies were again screened by immunoblottmg [15]. Two posture colonies were isolated. Crude cell extracts were prepared and analyzed for G T P cyclohydrolase 1 activity which was found about 100-fold mcreased as compared to the parent strain, JM83. Cell extracts were subjected to SDS electrophoresls followed by Western blotting (Fig. 1) Both clones showed strong tmmunoreactive bands corresponding to M r = 24000 which appeared identical with the wild-type enzyme. The plasmids obtained from the clones analyzed showed the presence of a 3.8 kb insert upon resmctton with BamHI. Plasmtd pCYH was used m subsequent work. A preliminary restrmtion map of the insert is shown in Ftg. 2. The msert contains no cutoog rotes for the following enzymes: Aval. Sacl, BanII, XbaI.
234 BH
I I t
lkb
P
B
I
I
i
Fig 2 Restncuonmap of the 38kb BamH[ insert containing the cloned GTP ¢yclohydrolase 1 gene Plasrmd pCYH was obtained by subclomngof one of the BamHI fragments from Agtll clone m pUCI3 Enzyme sites are indicated by a one letterdesignation(B, BamHI, P, /~stl,H, HmdlII) From 40 g of cells of E. colt JM83 pCYH, we could isolate 100 mg of pure GTP cyclohydrolas¢ I usmg the protocol of Yim and Brown with the modihcattong described under MATERIALS AND METHODS. The protein appeared identical with the wild type enzyme as judged by SDS gel electrophoresis. By comparison, 8 mg of enzyme were obtained from 500 g of E, cob wild type cells.
5. DISCUSSION The presence of a 3.8 kb BaraHl fragment of E. colt in a pUCI3 plasrmd leads to an apprmamately 100 fold increased level of GTP cyclohydrolase I activity in E, colt. The enzyme isolated from the plaslmd-carrymg strain has a subunit molecular weight of 24000 and appears identical with the wild type protein. Tins suggests that the 3.8 kb BamHI fragment harbors the entire structural gene for GTP ¢yclohydrolase I including the promoter and the termination signal. We cannot formally rule out the possibihty that the 3.8 kb fragment codes for an activator protein conducing to a dramatic increase In the expression of the chromosomal GTP cyclohydrolase I gene, but the hypothesis appears rather unlikely, since there is at present no evidence for regulauon of any type of GTP cyclohydrolase I in E. colt. The preparation of GTP cyclohydrolase I t s greatly facilitated by the increased enzyme levels contained in the plasmid harboring strains. There is no evidence for a negative effect of the increased enzyme level on the viabihty of the organism. More detailed studies of the structure and function of the enzyme should become pnssi-
ble by the improved availability of the protein. Moreover, the enzyme is a useful reagent for the preparation of dihydroneopt.erin 3'-tnphosphate for which there is increased demand as a consequence of the novel interest in the biosynthesis and ragulauon of unconjugated ptertns in mammals.
ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgememschaft and the Fond der Chemlschen lndustne. We thank Drs. F. GiStz and A. BiSck for helpful discussions.
REFERENCES [ll Brown, (3 M and Wdhamson, J M (1982)Adv Enzymol 53, 345-35L [2} B]au, N. and Nlederwleser, A (1985) J Chll Chem. CHIn.
BlOchem 23,169-176 [3] Ylm, J J and Brown, G M (1976) J Btol Chem 251, 5087-5094 [4] Ntcbol,C A, Smith, G K and l)ud~ D S (1985lAnnu Rev Blochern 54, 729-764, 151 Z~egler,I (1987)Naturwssenschaften 74, 563-572
[6] Schoedon, G.S, Redwelk, U and Curtius, H C. (1989) Eur J Bsochem 1"/8,627-634 [7i Young,R A and Davis,R W (1983)Scmnce222,778-782 [8] Yamsch-Perron, C. Vlelra,J. and Messing,J (1988)Gene 33,103-119. [9] Murray, N.E. Brammer, WJ and Murray, K (197"/) Melee Gen Genet 150,53-61 [10] Laemmh,U.K, (19'/0) Nature 227, 680-685 [11] Towbm, H., Staehhn, T and Gordon, J (1979)Proc Nat #.cad Sol USA 76, 4350-4354 [12] Mte~ndorL Re,, Percy, C and Young, R,A (1987) M¢lhOdsEnzymol 152,458-469 [131 Mamaus, T. Fntsch, E,F, and Sambn~ok, I (1982) Molecular clomng" A laboratory manual Cold Spnng Harbor Laboratory, New York [14] Huynh, TV, Young, R.A and Davis, R.W (1985) m DNA Cloning,'4ol 1 (Glover.D M, ed ), pp 49-78, IRL Press Lid,, Oxford 115l Hdfman, D,M, and Hughes, SH, (1987) Methods Enzymol 152,451-457