Increase of 10-deacetyl baccatin Ⅲ-10<i>β</i>-O-acetyl transferase activity with molecular docking simulation and site-directed mutation

OUYANG Pinglan; HUANG Jiajun; LIN Shuling; WEI Tao; LIN Junfang; GUO Liqiong; LIU Junfeng; LIU Qiqian

doi:10.7671/j.issn.1001-411X.2018.05.013

Journal of South China Agricultural University > 2018 > 39(5): 87-92. > DOI: 10.7671/j.issn.1001-411X.2018.05.013

OUYANG Pinglan, HUANG Jiajun, LIN Shuling, WEI Tao, LIN Junfang, GUO Liqiong, LIU Junfeng, LIU Qiqian. Increase of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase activity with molecular docking simulation and site-directed mutation[J]. Journal of South China Agricultural University, 2018, 39(5): 87-92. DOI: 10.7671/j.issn.1001-411X.2018.05.013

Citation:

PDF (849 KB)

Increase of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase activity with molecular docking simulation and site-directed mutation

OUYANG Pinglan^{1, 2,},
HUANG Jiajun^{1, 2},
LIN Shuling^{1, 2},
WEI Tao^{1, 2},
LIN Junfang^{1, 2, ,},
GUO Liqiong^{1, 2, ,},
LIU Junfeng¹,
LIU Qiqian¹

1.
College of Food Science, South China Agricultural University, Guangzhou 510642, China
2.
Research Center for Microecologics Engineering of Guangdong Province, Guangzhou 510640, China

More Information

Received Date: January 28, 2018
Available Online: May 17, 2023

Graphical Abstract

Abstract

Abstract

Objective
To increase the activity of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase (DBAT) and the efficiency of in vitro enzymatic synthesis of baccatin Ⅲ.

Method
Computer simulation of molecular docking between DBAT and its natural substrate acetyl CoA was performed. Three amino acid residues including C165W, N300I and F160C were chosen for site-directed mutation. The recombinant mutants were expressed in Escherichia coli and their enzymatic properties were determined by reactions in vitro.

Result
The relative molecular mass of each of the three mutant enzymes was 67 000, and the optimum reaction temperature was 32.5 ℃ which was consistant with that of the wild type enzyme (DBAT). The optimum reaction pH of DBAT^F160C or DBAT was 7.5, slightly higher than that of DBAT^C165W or DBAT^N300I (pH 7.0). Compared with DBAT, DBAT^C165W, DBAT^F160C and DBAT^N300I had 61.5%, 59.6% and 19.2% higher specific activities, 55.4%、35.1% and 2.9% higher catalytic efficiency, as well as higher K_m and v_max.

Conclusion
Hydrophobic mutations lead to significantly increased specific activities for mutant enzymes compared with the wild type enzyme. This study provides a basis for efficient in vitro enzymatic synthesis of baccatin Ⅲ, a direct precursor for chemical semi-synthesis of anti-cancer drug paclitaxel.
- 10-deacetyl baccatin III-10β-O-acetyl transferase,
- molecular docking,
- site-directed mutation,
- specific activity,
- enzymatic reaction,
- baccatin Ⅲ

FullText(HTML)

References (21)

References

[1]	FERLAY J, SOERJOMATARAM I, DIKSHIT R, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012[J]. Int J Cancer, 2015, 136(5): E359-E386.
[2]	邓立新. 抗癌新药紫杉醇简介[J]. 化学教学, 2006(1): 46-48.
[3]	WANI M C, TAYLOR H L, WALL M E, et al. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia[J]. J Am Chem Soc, 1971, 93(9): 2325-2357.
[4]	刘先芳, 梁敬钰, 孙建博. 紫杉醇: 具有里程碑意义的天然抗癌药物[J]. 世界科学技术: 中医药现代化, 2017, 19(6): 941-949.
[5]	周培华, 刘兰, 李干雄, 等. 紫杉醇的生产研究[J]. 轻工科技, 2012(5): 8-9.
[6]	KINGSTON D. The shape of things to come: Structural and synthetic studies of taxol and related compounds[J]. Phytochemistry, 2007, 68(14): 1844-1854.
[7]	WALKER K, CROTEAU R. Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli[J]. Proc Natl Acad Sci USA, 2000, 97(2): 583-587.
[8]	匡雪君, 王彩霞, 邹丽秋, 等. 紫杉醇生物合成途径及合成生物学研究进展[J]. 中国中药杂志, 2016, 41(22): 4144-4149.
[9]	LI Y, ZHANG G J, PFEIFER B A. Current and emerging options for taxol production[J]. Adv Biochem Eng Biotechnol, 2015, 148: 405-425.
[10]	杨春, 孔令义, 汪俊松. 加拿大红豆杉中紫杉烷类成分的研究进展及药源危机解决方法探讨[J]. 中国中药杂志, 2014, 39(11): 1961-1971.
[11]	LIU W C, GONG T, ZHU P. Advances in exploring alternative taxol sources[J]. RSC Advances, 2016, 6(54): 48800-48809.
[12]	郭斐. 利用半理性和理性策略对酶活性及手性选择性的设计[D]. 杭州: 浙江大学, 2015.
[13]	CHICA R A, DOUCET N, PELLETIER J N. Semi-rational approaches to engineering enzyme activity: Combining the benefits of directed evolution and rational design[J]. Curr Opin Biotechnol, 2005, 16(4): 378-384.
[14]	DILL K A. Dominant forces in protein folding[J]. Biochemistry, 1990, 29(31): 7133-7155.
[15]	王克夷. 疏水作用和蛋白质[J]. 生命的化学, 1999, 19(5): 233-235.
[16]	PETRUSKA J, SOWERS L, GOODMAN M. Comparison of nucleotide interactions in water, proteins, and vacuum: Model for DNA polymerase fidelity[J]. Proc Natl Acad Sci USA, 1986, 83(6): 1559-1562.
[17]	SHARMA B, KAUSHIK N, SINGH K, et al. Substitution of conserved hydrophobic residues in motifs B and C of HIV-1 RT alters the geometry of its catalytic pocket[J]. Biochemistry, 2002, 41(52): 15685-15697.
[18]	HAN Z, HAN S, ZHENG S, et al. Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface[J]. Appl Microbiol Biotechnol, 2009, 85(1): 117-126.
[19]	GALLARDO Ó, PASTOR FI, POLAINA J, et al. Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution[J]. J Biol Chem, 2010, 285(4): 2721-2733.
[20]	KOUDELAKOVA T, CHALOUPKOVA R, BREZOVSKY J, et al. Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel[J]. Angew Chem Int Ed Engl, 2013, 52(7): 1959-1963.
[21]	WAHAB R A, BASRI M, RAHMAN M B A, et al. Engineering catalytic efficiency of thermophilic lipase from Geobacillus zalihae by hydrophobic residue mutation near the catalytic pocket[J]. Adv Biosci Biotechnol, 2012, 3(2): 158-167.

Cited By

Get Citation

PDF

XML

Article views (1808) PDF downloads (1771)

Turn off MathJax

Article Contents

Abstract

References

Increase of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase activity with molecular docking simulation and site-directed mutation

Abstract

References

Catalog

Export File

Citation

Format

Content