A Molecular Ruler for Chain Elongation Catalyzed by Octaprenyl Pyrophosphate Synthase and Its Structure-Based Engineering To Produce Unprecedented Long Chain trans-Prenyl Products
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- 作者：Rey-Ting Guo ; Chih-Jung Kuo ; Tzu-Ping Ko ; Chia-Cheng Chou ; Po-Huang Liang ; and Andrew H.-J. Wang
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 22, 2004
- 年：2004
- 卷：43
- 期：24
- 页码：7678 - 7686
- 全文大小：587K
- 年卷期：v.43,no.24(June 22, 2004)
- ISSN：1520-4995
文摘
Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions offarnesyl pyrophosphate (FPP) with five molecules of isopentenyl pyrophosphate (IPP) to generate C40octaprenyl pyrophosphate (OPP) which constitutes the side chain of menaquinone. We have previouslyreported the X-ray structure of OPPs from Thermotoga maritima, which is composed entirely of 
-helicesjoined by connecting loops and is arranged with nine core helices around a large central cavity [Guo, R.T., Kuo, C. J., Ko, T. P., Chou, C. C., Shr, R. L., Liang, P. H., and Wang, A. H.-J. (2004) J. Biol. Chem.279, 4903-4912]. A76 and S77 are located on top of the active site close to where FPP is bound. A76Yand A76Y/S77F OPPs mutants produce C20, indicating that the substituted larger residues interfere withthe substrate chain elongation. Surprisingly, the A76Y/S77F mutant synthesizes a larger amount of C20than the A76Y mutant. In the crystal structure of the A76Y/S77F mutant, F77 is pushed away by Y76,thereby creating more space between those two large amino acids to accommodate the C20 product. Alarge F132 residue at the bottom of the tunnel-shaped active site serves as the "floor" and determines thefinal product chain length. The substitution of F132 with a small Ala, thereby removing the blockade, ledto the synthesis of a C50 product larger than that produced by the wild-type enzyme. On the basis of thestructure, we have sequentially mutated the large amino acids, including F132, L128, I123, and D62, toAla underneath the tunnel. The products of the F132A/L128A/I123A/D62A mutant reach C95, beyondthe largest chain length generated by all known trans-prenyltransferases. Further modifications of theenzyme reaction conditions, including new IPP derivatives, may allow the preparation of high-molecularweight polyprenyl products resembling the rubber molecule.
-helicesjoined by connecting loops and is arranged with nine core helices around a large central cavity [Guo, R.T., Kuo, C. J., Ko, T. P., Chou, C. C., Shr, R. L., Liang, P. H., and Wang, A. H.-J. (2004) J. Biol. Chem.279, 4903-4912]. A76 and S77 are located on top of the active site close to where FPP is bound. A76Yand A76Y/S77F OPPs mutants produce C20, indicating that the substituted larger residues interfere withthe substrate chain elongation. Surprisingly, the A76Y/S77F mutant synthesizes a larger amount of C20than the A76Y mutant. In the crystal structure of the A76Y/S77F mutant, F77 is pushed away by Y76,thereby creating more space between those two large amino acids to accommodate the C20 product. Alarge F132 residue at the bottom of the tunnel-shaped active site serves as the "floor" and determines thefinal product chain length. The substitution of F132 with a small Ala, thereby removing the blockade, ledto the synthesis of a C50 product larger than that produced by the wild-type enzyme. On the basis of thestructure, we have sequentially mutated the large amino acids, including F132, L128, I123, and D62, toAla underneath the tunnel. The products of the F132A/L128A/I123A/D62A mutant reach C95, beyondthe largest chain length generated by all known trans-prenyltransferases. Further modifications of theenzyme reaction conditions, including new IPP derivatives, may allow the preparation of high-molecularweight polyprenyl products resembling the rubber molecule.© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |