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Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Abstract

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50–52% identical to known higher plant cycloartenol synthases and 40–49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

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Abbreviations

A:

alanine

C:

cysteine

D:

aspartate

E:

glutamate

F:

phenylalanine

G:

glycine

H:

histidine

I:

isoleucine

K:

lysine

L:

leucine

M:

methionine

N:

asparagine

P:

proline

Q:

glutamine

R:

arginine

S:

serine

T:

threonine

V:

valine

W:

tryptophan

Y:

tyrosine

AaSHC:

Alicyclobacillus acidocaldarius squalene-hopene cyclase

cDNA:

complementary deoxyribonucleic acid

GC/MS:

gas chromatography/mass spectrometry

HSQC:

heteronuclear single quantum coherence

kbp:

kilobase pair

NMR:

nuclear magnetic resonance

PCR:

polymerase chain reaction

PEG:

polyethylene glycol

SC-UHET+gal:

synthetic complete medium lacking uracil with 2% galactose, 13 mg/L heme, 20 mg/L ergosterol, 0.5% Tween 80

ScERG7:

Saccharomyces cerevisiae lanosterol synthase

SC-UHET+glu:

synthetic complete medium lacking uracil with 2% glucose, 13 mg/L heme, 20 mg/L ergosterol, 0.5% Tween 80

SHC:

squalene-hopene cyclase

TLC:

thin-layer chromatography

YPDHET:

1% yeast extract, 2% peptone, 2% glucose, 13 mg/L heme, 20 mg/L ergosterol, and 0.5% Tween 80

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Correspondence to Seiichi P. T. Matsuda.

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Godzina, S.M., Lovato, M.A., Meyer, M.M. et al. Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA. Lipids 35, 249–255 (2000). https://doi.org/10.1007/s11745-000-0520-3

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  • DOI: https://doi.org/10.1007/s11745-000-0520-3

Keywords

  • Lanosterol
  • Dictyostelium Discoideum
  • Euglena Gracilis
  • Cycloartenol
  • Triterpene Alcohol