• Title/Summary/Keyword: Acyl protein thioesterase

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Biochemical Characteristics of a Palmitoyl Acyl Carrier Protein Thioesterase Purified from Iris pseudoacorus

  • Kang, Han-Chul;Hwang, Young-Soo
    • BMB Reports
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    • v.29 no.5
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    • pp.436-441
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    • 1996
  • The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

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Purification and Characterization of Myristoyl-Acyl Carrier Protein Thioesterase from Iris tectorum

  • Kang, Han-Chul;Cho, Kang-Jin;Hwang, Young-Soo
    • Applied Biological Chemistry
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    • v.41 no.4
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    • pp.235-240
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    • 1998
  • The myristoyl-acyl carrier protein (ACP) specific thioesterase from Iris tectorum was purified to a considerable homogeneity and characterized. The enzyme was eluted with a considerable stability by double-gradients using Triton X-100 and low ionic KCl or Na-phosphate through DEAE-52, Octyl-Sepharose, Q-Sepharose, and hydroxyapatite chromatoraphy. SDS-PAGE analysis showed a single band of 39 kDa. The native molecular weight was estimated to be 82 kDa by Sephacryl S-200 chromatography, indicating that the enzyme was a dimer. The thioesterase showed a chain-length specificity to myristoyl-ACP in preference to other-ACPs. The enzyme activity decreased by 1.0 mM myristate to about 27% of the original activity, whereas the remaining activity with decanoate was about 90%. The purified thioesterase was inhibited by myristoyl-CoA more than by myristate, suggesting that the myristoyl-AGP thiolesterase might be controlled by myristic acid and/or a subsequent product myristoyl-CoA. In addition, some biochemical characteristics of the enzyme were described.

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Overexpression of Cuphea viscosissima CvFatB4 enhances 16:0 fatty acid accumulation in Arabidopsis

  • Yeon, Jinouk;Park, Jong-Sug;Lee, Sang Ho;Lee, Kyeong-Ryeol;Yi, Hankuil
    • Journal of Plant Biotechnology
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    • v.46 no.4
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    • pp.282-290
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    • 2019
  • Cuphea viscosissima plants accumulate medium-chain fatty acids (MCFAs), i.e., those containing 8 ~ 14 carbons, in their seeds, in addition to the longer carbon chain fatty acids (≥16 carbons) found in a variety of plant species. Previous studies have reported the existence of three C. viscosissima MCFA-producing acyl-acyl carrier protein (ACP) thioesterases with different substrate specificities. In this study, CvFatB4, a novel cDNA clone encoding an acyl-ACP thioesterase (EC 3.1.2.14), was isolated from developing C. viscosissima seeds. Sequence alignment of the deduced amino acid sequence revealed that four catalytic residues for thioesterase activity are conserved and a putative N-terminal chloroplast transit peptide is present. Overexpression of CvFatB4 cDNA, which was under the control of the cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana led to an increase in 16:0 fatty acid (palmitate) levels in the seed oil at the expense of 18:1 and other non-MCFAs.

The Clinical and Pathologic Features according to Expression of Acyl Protein Thioesterase-1 (APT1) in Stage I Non-small Cell Lung Cancer (제1기 비소세포폐암에서 APT1 발현의 임상적 의미)

  • Shin, Jung-Ar;Lee, Chang-Ryul;Byun, Min-Kwang;Chang, Yoon-Soo;Kim, Se-Kyu;Chang, Joon;Ahn, Chul-Min;Kim, Hyung-Jung
    • Tuberculosis and Respiratory Diseases
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    • v.68 no.4
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    • pp.212-217
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    • 2010
  • Background: Acyl protein thioesterase-1 (APT1) is a cytosolic protein that may function in the depalmitoylation of numerous proteins, including the Ras family. However, the clinical role of depalmitoyl thioesterase in human cancer is not known. We evaluated the APT1 expression in lung cancer tissue and its clinicopathological findings according APT1 expression pattern. Methods: APT1 expression was examined by immunohistochemistry in the tumor tissue from 79 patients, who had undergone curative surgical removal of the primary lesion; all patients had been diagnosed with stage I non-small cell lung cancer between 1993 and 2004, at Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Results: The APT1 expression was seen in 50 out of 79 (63.3%) cases. The positive APT1 expression was significantly related with histologic subtype and T stage, but was not influenced by differentiation. The positive APT1 expression was not significantly related to patient age, gender, or smoking history. The median follow-up duration was 10.0 years; the 5-year survival rate was 71.0%. The positive APT1 expression group showed significantly worse overall survival and worse disease-free survival without statistical significance. Conclusion: We conclude that positive APT1 expression in stage I lung cancer after surgery is closely associated with overall survival. To evaluate APT1 as a prognostic marker in lung cancer, comprehensive studies on advanced stage cases are needed.

Cloning and Analysis of a Type II Polyketide Synthase Gene Cluster from Streptomyces toxytricini NRRL 15,443

  • Yoo An-Na;Demirev Atanas V.;Lee, Ji-Seon;Kim, Sang-Dal;Nam Doo-Hyun
    • Journal of Microbiology
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    • v.44 no.6
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    • pp.649-654
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    • 2006
  • A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), $\beta$-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.