Publications

• The cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae.

  Poklepovich T.J., Rinaldi M.A., Tomazic M.L., Favale N.O., Turkewitz A.P., Nudel C.B., Nusblat A.D.

  Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt ... >> More

  Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b(5), Cyt b(5) reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. << Less

  Steroids 77:1313-1320(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Partial purification and characterization of lathosterol 5-desaturase from rat liver microsomes.

  Honjo K., Ishibashi T., Imai Y.

  The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparati ... >> More

  The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol. << Less

  J. Biochem. 97:955-959(1985) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase.

  Arthington B.A., Bennett L.G., Skatrud P.L., Guynn C.J., Barbuch R.J., Ulbright C.E., Bard M.

  The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open read ... >> More

  The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function. << Less

  Gene 102:39-44(1991) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• THE INTERMEDIARY ROLE OF DELTA-5,7-CHOLESTADIEN-3-BETA-OL IN CHOLESTEROL BIOSYNTHESIS.

  DEMPSEY M.E., SEATON J.D., SCHROEPFER G.J. Jr., TROCKMAN R.W.

  J Biol Chem 239:1381-1387(1964) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Plant sterol biosynthesis: identification and characterization of higher plant delta 7-sterol C5(6)-desaturase.

  Taton M., Rahier A.

  Microsomes obtained from maize seedlings catalyzed the introduction of the delta5-bond into delta7-sterols to yield the corresponding delta 5,7-sterols. Enzymatic bioassay conditions have been developed for the first time for delta 7-sterol C5(6)-desaturase in photosynthetic organisms. The propert ... >> More

  Microsomes obtained from maize seedlings catalyzed the introduction of the delta5-bond into delta7-sterols to yield the corresponding delta 5,7-sterols. Enzymatic bioassay conditions have been developed for the first time for delta 7-sterol C5(6)-desaturase in photosynthetic organisms. The properties of the microsomal system have been studied and the kinetics of the desaturation reaction has been established. The desaturation reaction requires molecular oxygen and NADH. Coenzyme efficiency studies indicate that NADH is more efficient that NADPH and that in the presence of NADH, NAD+ stimulates the desaturation process but cannot sustain the reaction by itself. The desaturation is strongly inhibited by cyanide, is sensitive to 1,10-phenanthroline and to salicylhydroxamic acid, but is insensitive to carbon monoxide, suggesting the involvement of a metal ion, presumably iron, in an enzyme-bound form in the desaturating system. From a series of incubations with delta 7-sterols and other sterol analogs, the substrate specificity for desaturation was determined. Our data indicate the substrate selectivity of the C5(6)-desaturation for 4-desmethyl-delta 7-sterols. Moreover, the results show that specificity of maize C5(6)-desaturase favored delta 7-sterols possessing a C24-methylene or ethylidene substituent compared to 24-ethyl-substituted delta 7-sterols. Finally, the results demonstrate directly that during plant sterol synthesis the delta 5-bond is introduced via the sequence delta 7-sterol-->delta 5,7-sterol-->delta 5-sterol. << Less

  Arch Biochem Biophys 325:279-288(1996) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Role of highly conserved residues in the reaction catalyzed by recombinant Delta7-sterol-C5(6)-desaturase studied by site-directed mutagenesis.

  Taton M., Husselstein T., Benveniste P., Rahier A.

  The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol ... >> More

  The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols. In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations. One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs. Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues. Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type. Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects. One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations [Shanklin et al. (1994) Biochemistry 33, 12787-12794]. Another group of mutants was affected in residue 114 based on previous in vivo observations in A. thaliana indicating that mutant T114I was deficient in 5-DES activity. We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency. Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme. Consequently, V/K for T114S was 38-fold higher than that for T114I. The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES. << Less

  Biochemistry 39:701-711(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Temperature-induced differential kinetic properties between an initial burst and the following steady state in membrane-bound enzymes: studies on lathosterol 5-desaturase.

  Nishino H., Nakaya J., Nishi S., Kurosawa T., Ishibashi T.

  The NADH-dependent lathosterol 5-desaturation reaction that forms 7-dehydrocholesterol is biphasic, an initial burst followed by steady state. The steady-state phase is slower than the burst phase, because the latter diffusion of the lathosterol substrate within the microsomal membrane must occur ... >> More

  The NADH-dependent lathosterol 5-desaturation reaction that forms 7-dehydrocholesterol is biphasic, an initial burst followed by steady state. The steady-state phase is slower than the burst phase, because the latter diffusion of the lathosterol substrate within the microsomal membrane must occur before the next reaction can take place [Y. Takakuwa, H. Nishino, Y. Ishibe, and T. Ishibashi (1994) J. Biol. Chem. 269, 27889-27893]. In the present study, changes in the structure and function of the membrane were examined by measurement of the Arrhenius activation energy of lathosterol 5-desaturase at various temperatures between 2 and 45 degrees C. At the burst phase, there was a lack of discontinuity in the Arrhenius plots at the presumed phase transition temperature for the microsomal membrane. However, the plots of the activities of the steady state showed breaks at around 17 and 32 degrees C. It was concluded that phospholipid phase transition affects the steady-state phase but not the burst phase. Furthermore, treatment of microsomes with low concentrations of deoxycholate, known to perturb the membrane integrity, resulted in a break of the activation energy of the burst phase. These results have revealed further evidence for our previous model suggesting interaction between the substrate and enzyme within the microsomal membrane via lateral diffusion. << Less

  Arch. Biochem. Biophys. 339:298-304(1997) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• cDNA cloning of the mammalian sterol C5-desaturase and the expression in yeast mutant.

  Nishi S., Nishino H., Ishibashi T.

  Sterol C5-desaturase (SC5D) cDNA is an enzyme that catalyzes the dehydrogenation to introduce C5-6 double bond into lathosterol in cholesterol biosynthesis. We have isolated and sequenced the cDNA clones encoding human and mouse SC5D. The functional complementation of a defective yeast mutant prov ... >> More

  Sterol C5-desaturase (SC5D) cDNA is an enzyme that catalyzes the dehydrogenation to introduce C5-6 double bond into lathosterol in cholesterol biosynthesis. We have isolated and sequenced the cDNA clones encoding human and mouse SC5D. The functional complementation of a defective yeast mutant proves that the human and mouse cDNA clones encode SC5D. Mammalian SC5D was presumed as an integral membrane protein containing histidine residues conserved also in yeasts and plant. << Less

  Biochim. Biophys. Acta 1490:106-108(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.