Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, 5800 Río Cuarto, Córdoba, Argentina1
Departamento de Genética, Universidad de Sevilla, Sevilla, Spain2
Author for correspondence: Carlos E. Domenech. Tel: +54 58 676114. Fax: +54 58 676232. e-mail: cdomenech{at}exa.unrc.edu.ar
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ABSTRACT |
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Keywords: lovastatin, terpenoids, gibberellins, HMG-CoA reductase, Gibberella fujikuroi
Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A
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INTRODUCTION |
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All natural terpenoids are derived from geranyl diphosphate (Mende et al., 1997 ). The sterols, gibberellins and carotenoids of G. fujikuroi are synthesized through a pathway that includes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and D-mevalonate (Coolbaugh, 1983
; Domenech et al., 1996
). These intermediaries are not used in an alternative pathway as found in other organisms, which starts with glyceraldehyde 3-phosphate and pyruvate (Rohmer et al., 1993
).
Labelling experiments led to the conclusion that the pathways for different terpenoids, including their common sections, must occur in different cellular compartments. This is the case for carotene and sterol biosyntheses in Phycomyces blakesleeanus (Bejarano & Cerdá-Olmedo, 1992 ), and carotenoid, sterol and gibberellin biosyntheses in G. fujikuroi (Domenech et al., 1996
). Terpenoid biosynthesis in different compartments was also shown in animals (Biardi & Krisans, 1996
) and, as metabolic channels, in plants (Chappell, 1995b
).
HMG-CoA reductase (EC 1 . 1 . 1 . 34) catalyses the conversion of HMG-CoA to mevalonate. Genes for this enzyme have been isolated from several organisms. All of them contain a highly conserved, hydrophilic catalytic domain and a variable number of less conserved transmembrane domains (Hampton et al., 1996 ). Whereas in animals there appears to be only one gene for HMG-CoA reductase, two or more genes have been found in Saccharomyces cerevisiae and in different plants (Basson et al., 1988
; Chappell, 1995a
). A gene from G. fujikuroi can be readily isolated using degenerate oligonucleotides with sequences that are highly conserved in all homologous genes (Corrochano & Avalos, 1992
; Woitek et al., 1997
).
A powerful inhibitor of this enzyme is a compound now most frequently called lovastatin, previously known as monacolin K when isolated from Monascus ruber and mevinolin when isolated from Aspergillus terreus (Endo & Hasumi, 1997 ). Lovastatin inhibits growth in many organisms, often at very low concentrations, presumably because of the inhibition of the synthesis of essential terpenoids (Florin-Christensen et al., 1990
; Lam & Doolittle, 1992
; Haag et al., 1994
; Morehead et al., 1995
).
In this paper we show that while lovastatin did not significantly affect the growth and production of sterols and carotenoids in G. fujikuroi, it did inhibit its gibberellin production. Our results support the existence of independent compartments for the biosynthesis of various terpenoids in this fungus.
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METHODS |
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Biochemical analyses.
Carotenoids were extracted with acetone and the dried extracts were redissolved in n-hexane for spectrophotometry. The total amounts of coloured carotenoids (Fernández-Martín et al., 1995 ) or ergosterol (Domenech et al., 1996
) were estimated from absorbance determinations. The total gibberellin concentrations in culture media were estimated fluorometrically (Candau et al., 1991
) after removal of mycelium by filtration.
To assay HMG-CoA reductase, washed mycelium was frozen in liquid nitrogen, ground in a mortar, and extracted by suspension in HEPES/sucrose/NaCl/EDTA/EGTA buffer, pH 7·4, plus protease inhibitors as described by Peña-Díaz et al. (1997) . The lysate was then cleared by centrifugation at 1000 g for 2 min. HMG-CoA reductase was assayed by utilizing 2550 µg protein from the supernatant and DL-3-hydroxy-3-methyl-[3-14C]glutaryl-CoA (Amersham International) as substrate as described by Shapiro et al. (1974)
with the modification of Peña-Díaz et al. (1997)
. Each sample contained 0·4 mM of the radioactive substrate (specific radioactivity: 2550 d.p.m. nmol-1), 25 mM EDTA, 1·6 mM DTT, 30 mM glucose 6-phosphate, 3 mM NADP, 50 milliunits glucose-6-phosphate dehydrogenase in a final volume of 75 µl. Under the assay conditions described the determinations were linear up to at least 30 min incubation. To study the effect of lovastatin in vitro, it was added, as the sodium salt, to cell-free extracts at concentrations indicated in the text or figures. The activity of HMG-CoA reductase was expressed as pmol HMG-CoA converted to mevalonate min-1 (mg protein)-1. Protein was determined as described by Bradford (1976)
, using BSA as standard. The analytical results are the mean and its standard error from three independent experiments.
Genomic DNA isolation.
Genomic DNA was isolated following a modification of the method described by Sherman et al. (1986) . Mycelium was obtained by filtration of a culture of 106 spores incubated for 2 d at 30 °C in an orbital shaker in 200 ml minimal medium (Avalos et al., 1985
) supplemented with 1 g yeast extract l-1. The mycelial pad was washed with water, dried with filter paper, covered with liquid nitrogen in a precooled mortar and quickly ground to a fine powder. The latter was mixed immediately with 7·5 ml Tris/EDTA (50 mM Tris, 20 mM EDTA, pH 7·5) and 0·5 ml SDS (100 g l-1) and heated at 65 °C for 30 min. It was then mixed with 2·3 ml 5 M potassium acetate, maintained at 4 °C for 3060 min, and centrifuged at 9000 g for 10 min. The supernatant was filtered through a gauze, mixed with 2 vols ethanol, and incubated overnight at 4 °C before centrifugation at 9000 g for 10 min. The pellet was washed twice with ethanol (700 ml l-1), suspended in 3 ml Tris/EDTA, and treated with 15 µl bovine pancreas ribonuclease A (10 g l-1) for 30 min at 37 °C. The sample was mixed with 2 ml phenol/chloroform/isoamyl alcohol (25:24:1, by vol.) to stop digestion and centrifuged at 3500 g for 5 min. The water fraction was washed twice with chloroform/isoamyl alcohol (15:1, v/v), transferred to a new clean tube, mixed with 1 vol. 2-propanol, and centrifuged at 1000 g for 5 min. The precipitate was washed twice with ethanol (700 ml l-1), dried and resuspended in 0·5 ml Tris/EDTA solution.
Southern hybridization and PCR replication.
Genomic DNA (approx. 5 µg) was digested overnight with the indicated restriction enzymes. It was then separated by electrophoresis in an agarose gel (1%), and transferred to a nylon membrane (Hybond; Amersham). Hybridization was carried out with a 318 bp fragment of the G. fujikuroi gene for HMG-CoA reductase, obtained by in vitro PCR replication of genomic DNA using the primers described by Corrochano & Avalos (1992) , labelled with digoxigenin-11-dUTP. Detection was carried out with a chemiluminescent substrate (CSPD) following the manufacturers recommendations (Boehringer Mannheim). Hybridization and washing were carried out at 65 °C. For other DNA manipulations and technical details see Sambrook et al. (1989)
.
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RESULTS |
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DISCUSSION |
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The second point supporting the idea that the enzyme is not freely accessed by its inhibitor are the results obtained by Southern hybridization and PCR amplification. The results of both types of experiments led to conclusions very similar to those reported by Woitek et al. (1997) for different strains of G. fujikuroi. HMG-CoA reductase appears to be the product of a single gene. The restriction pattern of the PCR product is especially informative: our mixture of primer oligonucleotides should have allowed detection of all known genes for HMG-CoA reductases (Corrochano & Avalos, 1992
), but amplification resulted in only one DNA sequence that appears to be unique. Although a single hybridizing band in a genomic Southern blot may constitute proof that a gene is present in one copy per haploid genome, our results do not rule out the fact that a tandemly repeated gene array would give the same pattern of hybridization.
An independent explanation would be that lovastatin inhibits not only HMG-CoA reductase, but also a step specific for gibberellin biosynthesis. This would require different compartmentation, with only the latter step accessible to lovastatin in vivo. The normal gibberellin biosynthesis in the presence of lovastatin and mevalonate suggests that gibberellins are produced freely from mevalonate in the presence of lovastatin. Mevalonate should have a regulatory effect of its own that leads to a total blockage of gibberellin production when present initially in the culture media (Domenech et al., 1996 ); for this reason mevalonate was added to 3-d-old cultures.
Alternatively, G. fujikuroi could possess two HMG-CoA reductases. The essential terpenoids and the carotenoids would be made by an enzyme belonging to a wholly new sequence class, resistant to lovastatin, and undetectable in the in vitro assay. This hypothesis, however, lacks experimental support and is contrary to observations of enzymes from numerous organisms.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 December 1999;
revised 24 June 1999;
accepted 5 July 1999.