|Table of Contents|

Molecular Cloning and Heterologous Expression of Putative Strictosidine Synthases from Arabidopsis thaliana(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

Issue:
2013 02
Page:
224-230
Research Field:
Articles
Publishing date:

Info

Title:
Molecular Cloning and Heterologous Expression of Putative Strictosidine Synthases from Arabidopsis thaliana
Author(s):
MA Lei ZHANG Xiang TIAN Yongqiang ZHANG Guolin LUO Yinggang
(1College of Chemical Engineering, Sichuan University, Chengdu, 610065, China)
(2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)
Keywords:
terpenoid indole alkaloids strictosidine synthase Arabidopsis thaliana RT-PCR PCR heterologous expression
CLC:
Q946.88 : Q78
PACS:
DOI:
10.3724/SP.J.1145.2013.00224
DocumentCode:

Abstract:
No complex alkaloids were proved to accumulate in Arabidopsis thaliana. However, many genes in the genome of A. thaliana were annotated as homologues to the genes involved in the biosynthetic pathway of terpenoid indole alkaloids (TIAs), for instance, 15 genes were annotated as strictosidine synthase (STR). To clarify the function of these genes, we selected 5 putative AtSTRs out of 15 genes on the basis of bioinformatic analysis of known functional RsSTR and AtSTRs. The five putative AtSTR genes were obtained by RT-PCR using total RNA as template. The plasmid containing AtSTRs were constructed, screened by the combination of X-gal and IPTG, enzymtic digestion and PCR. The sequences of these AtSTRs were confirmed by DNA sequencing. The expression plasmids were constructed, sequenced, and transformed into Escherichia coli BL21 (DE3) for protein overexpression. The fusion proteins were soluble and purified by nickel ion affinity chromatography. The putative catalytic activity of AtSTRs was assayed at four levels, including whole cells, cell lysates, supernatant, and purified enzyme, by adding the native substrates tryptamine and secologanin to the reaction mixture. No desired or new products were observed in the reaction mixture by HPLC-DAD detection, which suggested that more work is needed to clarify the possible function of these genes. Fig 8, Tab 3, Ref 22

References

1 O’Connor SE, Maresh JJ. Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat Prod Rep, 2006, 23 (4): 532-547
2 Gobbi PG, Broglia C, Merli F, Dell’Olio M, Stelitano C, Iannitto E, Federico M, Berte R, Luisi D, Molica S, Cavalli C, Dezza L, Ascari E. Vinblastine, bleomycin, and methotrexate chemotherapy plus irradiation for patients with early-stage, favorable Hodgkin lymphoma: the experience of the Gruppo Italiano Studio Linfomi. Cancer, 2003, 98 (11): 2393-2401
3 Qweider M, Gilsbach JM, Rohde V. Inadvertent intrathecal vincristine administration: a neurosurgical emergency. J Neurosurg Spine, 2007, 6 (3): 280-283
4 Geddes ER, Cohen PR. Antineoplastic agent-associated serpentine supravenous hyperpigmentation: superficial venous system hyperpigmentation following intravenous chemotherapy. South Med J, 2010, 103 (3): 231-235
5 Baumeister AA, Hawkins MF, Uzelac SM. The myth of reserpine-induced depression: role in the historical development of the monoamine hypothesis. J Hist Neurosci, 2003, 12 (2): 207-220
6 Brembilla-Perrot B, al Ammar A, Terrier de la Chaise A, Cherrier F, Pernot C. Arrhythmogenic effect of ajmaline on the atrial level. Arch Mal Coeur Vaiss, 1989, 82 (12): 1983-1990
7 Li S, Long J, Ma Z, Xu Z, Li J, Zhang Z. Assessment of the therapeutic activity of a combination of almitrine and raubasine on functional rehabilitation following ischaemic stroke. Curr Med Res Opin, 2004, 20 (3): 409-415
8 Maresh JJ, Giddings LA, Friedrich A, Loris EA, Panjikar S, Trout BL, Stockigt J, Peters B, O’Connor SE. Strictosidine synthase: mechanism of a Pictet-Spengler catalyzing enzyme. J Am Chem Soc, 2008, 130 (2): 710-723
9 Brown RT, Smith GN, Staplefo.Ks. Biosynthesis of strictosidine. Tetrahedron Lett, 1968, 41: 4349
10 Leveque D, Jehl F. Molecular pharmacokinetics of catharanthus (vinca) alkaloids. J Clin Pharmacol, 2007, 47 (5): 579-588
11 Mizukami H, Nordlov H, Lee SL, Scott AI. Purification and properties of strictosidine synthetase (an enzyme condensing tryptamine and secologanin) from Catharanthus roseus cultured cells. Am Chem Soc, 1979, 18 (17): 4
12 Treimer JF, Zenk MH. Purification and properties of strictosidine synthase, the key enzyme in indole alkaloid formation. Eur J Biochem, 1979, 101 (1): 225-233
13 Kutchan TM. Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli. FEBS Lett, 1989, 257 (1): 127-130
14 Kutchan TM, Hampp N, Lottspeich F, Beyreuther K, Zenk MH. The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli. FEBS Lett, 1988, 237 (1/2): 40-44
15 Kutchan TM. Strictosidine - from alkaloid to enzyme to gene. Phytochemistry, 1993, 32 (3): 493-505
16 Ma X, Panjikar S, Koepke J, Loris E, Stockigt J. The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed β-propeller fold in plant proteins. Plant Cell, 2006, 18 (4): 907-920
17 Meinke DW, Cherry JM, Dean C, Rounsley SD, Koornneef M. Arabidopsis thaliana: a model plant for genome analysis. Science, 1998, 282 (5389): 662, 679-682
18 Meyerowitz EM. Arabidopsis thaliana. Annu Rev Genet, 1987, 21: 93-111
19 Federspiel N. Deciphering a weed. Genomic sequencing of Arabidopsis. Plant Physiol, 2000, 124 (4): 1456-1459
20 Facchini PJ, Bird DA, St-Pierre B. Can Arabidopsis make complex alkaloids? Trends Plant Sci, 2004, 9 (3): 116-122
21 Sohani MM, Schenk PM, Schultz CJ, Schmidt O. Phylogenetic and transcriptional analysis of a strictosidine synthase-like gene family in Arabidopsis thaliana reveals involvement in plant defence responses. Plant Biol (Stuttg), 2009, 11 (1): 105-117
22 Stockigt J, Hammes B, Ruppert M. Construction and expression of a dual vector for chemo-enzymatic synthesis of plant indole alkaloids in Escherichia coli. Nat Prod Res, 2010, 24 (8): 759-766

Memo

Memo:
-
Last Update: 2013-05-03