Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua

Gene, 172 (1996) 207-209 0 1996 Elsevier Science B.V. All rights reserved.

207

0378-l 119/96/$15.00

GENE 09652

Short Communications

Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua (Artemisinin; cloned gene expression; FPP synthase; isoprenoid biosynthesis; medicinal plant; sesquiterpenoid)

Yasuhiko Matsushita, WonKyung Kang* and Barry V. Charlwood Division ofLife Sciences, King’s College London, London, WS 7AH, UK Received by J.R. Kinghorn:

10 October

1995; Revised/Accepted:

22 November

1995; Received at publishers:

22 January

1996

SUMMARY

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39 420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.

INTRODUCTION

Farnesyl diphosphate (FPP) synthase (FPPS) [dimethylallyl transferase (EC 2.5.1.1) and geranyl transferase (EC 2.5.1.10)] is a l’-4 prenyltransferase which catalyzes two consective condensations of isopentenyl diphosphate (IPP) with the allylic diphosphates, dimethylallyl diphosphate (DMAPP) and the resultant geranyl diphosphate (GPP). The ultimate product of these two reactions, FPP, Correspondence to: Dr. Y. Matsushita, College

London,

(+44-171)

Campden

333-4296;

Division

Hill Road,

Fax (+44-171)

of Life Sciences, King’s

London

W8 7AH,

UK. Tel.

333-4500,

e-mail: [email protected] * Present

address:

Consort

Road,

Abbreviations:

Department

London

of Biology,

Imperial

SW7 2BB, UK. Tel. (+44-171)

College,

Prince

594-5374.

aa, amino acid(s); Aa, Artemisia annua; Art, artemisinin;

.4t, Arabidopsis thaliana; bp, base pair(s); cDNA, DNA complementary to RNA; DMAPP, dimethylallyl diphosphate; EC, Escherichia coli; FPP, farnesyl

diphosphate;

diphosphate; phosphate;

FPPS,

GGPPS, IPP,

FPP

GGPP

isopentenyl

synthase(s); synthase(s);

diphosphate;

GGPP, GPP, IPTG,

geranylgeranyl geranyl

di-

isopropyl-8-w

thiogalactopyranoside; kb, kilobase or 1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction; SC, Saccharomyces cereuisiae; TLC, thin-layer chromatography; X, any aa; [I, denotes plasmid-carrier state. SSDI 0378-l

119(96)00054-6

is utilized in the biosynthesis of sterols, dolichols, mitochondrial electron transfer chain components, prenylated proteins and a wide range of sesquiterpenoids including phytoalexins (Bach, 1995). Genes encoding FPPS have been cloned from rat (Clarke et al., 1987), human (Sheares et al., 1989), Saccharomyces cerevisiae (SC) (Anderson et al., 1989) and more recently from plants such as Arabidopsis thaliana (At) (Delourme et al., 1994), Lupinus albus (Attucci et al., 1995) and Zea mays (GenBank accession No. L39789). Artemisia annua (Au), a medicinal plant originally native to Eastern Europe and China, produces a sesquiterpenoid endoperoxide called artemisinin (Art) which accumulates in the leaves and has antimalarial activity (Meshnick, 1994). We are interested in increasing the Art accumulation in Aa by genetic manipulation of the enzymes involved in the biosynthetic pathway, because of the medicinal value of Art. Since FPPS produces the 15-carbon product, FPP, which is utilized in the biosynthesis of the 15-carbon Art, genetic manipulation of this enzyme may affect the accumulation of Art. As a first step in the understanding and the manipulation of the pathway to the isoprenoids, particularly to Art, we have

208 isolated the Aa cDNA for FPPS. In this paper, we report the isolation and characterization of a cDNA for FPPS from a medical plant and the enzyme activities of the products expressed in bacteria.

EXPERIMENTAL

AND DISCUSSION

(a) Cloning of cDNAs encoding FPPS cDNA library was constructed from poly(A)+RNAs extracted from leaves of Aa using hUni-ZAP XR vector (Stratagene, La Jolla, CA, USA). In order to isolate the Au cDNAs for FPPS, we first designed two oligodeoxyribonucleotide primers corresponding to nt 337 to 356 and 771 to 752 of the At cDNA for FPPS (Delourme et al., 1994) and used them for PCR on the phage solution of the Aa cDNA library. Sequence analysis of the resultant 435-bp fragment showed that the deduced aa sequence was highly similar (85% identical) to that of At FPPS. Therefore, we used the 435-bp fragment as a probe to screen 1 x lo5 plaques of the Au cDNA library. Among several positive signals detected, five were selected and the phage clones then converted into plasmids (pAFPS1 to pAFPS5) in Escherichia coli (EC). pAFPS1 plasmid was found to contain the longest cDNA insert. The partial sequences of pAFPS3, pAFPS4 and pAFPS5 plasmids were determined from both ends of the cDNA inserts. The complete sequences of the cDNA inserts of pAFPS1 and pAFPS2 plasmids were obtained from both directions by sequencing some subcloned internal fragments and exonuclease III deletion derivatives of the cDNAs. The nt sequences of the pAFPS2 to pAFPS5 plasmids were completely identical to those of pAFPS1

over the portions that had been sequenced though the plasmids had different 5’ and 3’ ends of the cDNA inserts (Fig. 1). (b) Analysis of cDNA and deduced aa sequences Sequencing of pAFPS1 cDNA revealed a 343-aa product of 39 420 Da. The FPSl protein presents 76, 84 and 72% identity with those from plants, At, Lupinus albus and Zea mays, respectivly. The FPSl protein also have high similarity to those from SC (51% identical), rat (46%) and human (45%). The two domains, domain I ([ LIVM] [ LIVM] XDDXXDXXXXRRG) and domain II ([LIVMFY]GXXFQ[LIVM]XDD[LIVMFY]X[DN]) conserved among all polyprenyl synthases (Ashby and Edwards, 1990; Math et al., 1992) including FPPS, geranylgeranyl diphosphate (GGPP) synthases (GGPPS) and hexaprenyl diphosphate synthases were found in the FPS 1 protein (Fig. 1). Hydropathic analysis of FPS 1 protein showed similar patterns to those from At, yeast and mammals (data not shown). These characterics predicted from the deduced FPPS proteins suggest the conservation of their structures in eukaryotes. (c) Confirmation of the FPPS activity pAFPS2filB plasmid was constructed from pAFPS2 to adjust the reading frame of FPSl protein in the cDNA insert to that of P-galactosidase peptide in the vector. EC cells harbouring a plasmid pBluescript SK(-) (no insert), pAFPS2 (out-of-frame) or pAFPS2filB (in-frame) were induced with IPTG, and the cell extracts were analysed for FPPS activity. As shown in Table I, extracts from EC[ pAFPS2filBl (in-frame) afforded easily measureable levels of FPPS activity whilst Ec [ pBluescript SK( -)] (no

Fig. 1. Nucleotide sequences of pAFPS1 cDNA encoding Au FPPS. The cDNA was cloned by screening an Aa cDNA library with a PCR-derived probe and sequenced as described in section (a). The positions of 5’ and 3’ ends of the cDNAs of pAFPS2 to pAFPS5 plasmids are indicated by arrows with the plasmid number. The putative polyadenylation signal is underlined. The conserved Edwards, 1990; Math et al., 1992) are boxed. The sequence has been submitted to the GenBank/EMBL

aa residues in domain I and II (Ashby and Data Bank with accession No. U36376.

209 TABLE I FPPS activity in EC extractsa Plasmid

Encoded protein

DMAPPb

GPPb

Specific activity’ [nmol/min/mg of protein]

Standard deviation

pBluescript SK(-) pAFPS2 pAFPS2filB pAFPS2filB pAFPS2filB

none none FPSl FPSl FPSl

_ _ _

+ + + _

0.95 1.4 37 21 0.86

0.087 0.083 0.92 0.62 0.061

+ -

“FPPS activities were measured in EC cell extracts harbouring plasmids as indicated. Plasmid pAFPS2filB was constructed from pAFPS2 by digestion with BarnHI, filling-in and re-ligation. Cell extracts were prepared from EC XLl-Blue[pBluescript SK(-)], [pAFPS2] or [pAFPS2filB] as described by Sheares et al. (1989) except that cell disruption was carried out by sonication (MSE ultrasonic disintegrator at maximum power for 2 x 1 min bursts on an ice-water bath). Protein concentrations of the extracts were determined using Protein assay kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as standards. b FPPS assays were carried out in the presence (+) and absence (-) of DMAPP and GPP as described by Ogura et al. (1985) except that the reaction volume was scaled down to 100 u1 and lul of [ l-i4C]IPP (1.85 kBq, 2.11 GBq/mmol) was used for each reaction at 37°C for 5 min. ’ Specific FPPS activity of extracts was expressed as the formation of nmol of FPP/min/mg of protein with standard deviations. Each value of the specific activity represents the mean of those derived from three independent experiments.

insert) and EC[ pAFPS2] (out-of-frame) gave rise to little detectable catalytic activity. The observed activity was dependent on the addition of DMAPP or GPP to the reaction mixture since assays carried out in the absense of these allylic diphosphates showed little activity. Further analysis of the reaction products by TLC confirmed that the ultimate product derived from the extracts of Ec[pAFPS2filB] was FPP (data not shown). In addition, the cell extracts of pAFPS2filB plasmid were found to have no activity to convert FPP with IPP into GGPP in the presence of Mn ” (data not shown). Therefore, the protein encoded by the cDNA have activity for FPPS but not for GGPPS. These data confirm the identity of the cDNA as encoding Au FPPS.

ACKNOWLEDGEMENTS

We are grateful to Dr. S. Coomber for helpful discussions during this work and to Dr. P. Cunningham for his advice on computer analysis of DNA and protein sequences. We thank the Biotechnology and Biological Science Research Council for providing a postdoctoral fellowship (Y.M.) and for financial support.

REFERENCES Anderson, M.S., Yarger, J.G., Burck, C.L. and Poulter, C.D.: Farnesyl diphosphate synthase: molecular cloning, sequence, and expression

of an essential gene from Saccharomyces cereuisiae. J. Biol. Chem. 264 (1989) 19176-19184. Ashby, M.N. and Edwards, P.A.: Elucidation of the deficiency in two yeast coenzyme Q mutants: characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J. Biol. Chem. 265 (1990) 13157-13164. Attucci, S., Aitken, S.M., Ibrahim, R.K. and Gulick, P.J.: A cDNA encoding farnesyl pyrophosphate synthase in white lupine. Plant Physiol. 108 (1995) 835-836. Bach, T.J.: Some new aspects of isoprenoid biosynthesis in plants: a review. Lipids 30 (1995) 191-202. Clarke, C.F., Tanaka, R.D., Svenson, K., Wamsley, M., Fogelman, A.M. and Edwards, P.A.: Molecular cloning and sequence of a choresterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway. Mol. Cell Biol. 7 (1987) 3138-3146. Delourme, D., Lacroute, F. and Karst, F.: Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast. Plant Mol. Biol. 26 (1994) 1867-1873. Math, S.K., Hearst, J.E. and Poulter, C.D.: The ctrE in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc. Natl. Acad. Sci. USA 89 (1992) 6761-6764. Meshnick, S.R.: The mode of action of antimalarial endoperoxides. Trans. R. Sot. Trop. Med. Hyg. 88s (1994) 31-32. Ogura, K., Nishino, T., Shinka, T. and Seto, S.: Prenyltransferases of pumpkin fruit. Methods Enzymol. 110 (1985) 167-171. Sheares, B.T., White, S.S., Molowa, D.T., Chan, K., Ding, V.D.-H., Kroon, P.A., Bostedor, R.G. and Karkas, J.D.: Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells. Biochemistry 28 (1989) 8129-8135.