Deciphering the Key Loop: Enhancing l -Threonine Transaldolase's Catalytic Potential
文献类型：期刊论文
| 作者 | Xi, Zhiwen3,4; Rao, Jingxin2,3; Zhang, Xinyi4; Liu, Zhiyong3,4; Zheng, Mingyue2 ; Li, Lihong3,4; Zhang, Wenchi1; Xu, Yan3,4; Zhang, Rongzhen3,4
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| 刊名 | ACS CATALYSIS
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| 出版日期 | 2024-06-26 |
| 页码 | 13 |
| 关键词 | l-threonine transaldolase beta-hydroxy-alpha-aminoacids Loop engineering computational analysis byproduct elimination system |
| ISSN号 | 2155-5435 |
| DOI | 10.1021/acscatal.4c02049 |
| 通讯作者 | Zhang, Rongzhen(rzzhang@jiangnan.edu.cn) |
| 英文摘要 | l-Threonine transaldolase (LTTA) is an attractive biocatalyst because of its potential diastereoselectivity in the synthesis of beta-hydroxy-alpha-amino acids (beta HAAs). However, prospective development of LTTA has been hampered by its low activity. Here, a combination of techniques involving structural comparison, computational analysis, Loop deletion, and alanine scanning was used to identify a key Loop region (Loop 1) regulating the catalytic ability of Chitiniphilus shinanonensis LTTA (CsLTTA). Saturation mutagenesis and iterative saturation mutagenesis at the hot spots in Loop 1 were performed, and the best variant containing an F70T/C57Q/Y69T (TQT) triple mutation was screened. The diastereoisomer excess (de) produced by the TQT variant (95.4%(syn)) was greater than that produced by the wild-type (WT) enzyme (75.2%(syn) ), and the catalytic efficiency (k(cat)/K-m) of the TQT variant was four times higher than that of the wild-type enzyme. Molecular dynamics simulations, metadynamics simulations, and CAVER analysis revealed the critical role of the Loop 1 structure in regulating the hydrogen bond network and thus reshaping the active-site pocket to control the syn-tunnel direction. Further engineering of Loop 1 in ObiH, an LTTA responsible for obafluorin biosynthesis, resulted in the development of the F70T-C57Q-H69T (ObiH-TQT) variant producing a de of 97% syn . Using the ObiH-TQT variant for kilogram-scale synthesis of l-syn-p-methylsulfonylphenylserine, coupled with acetaldehyde elimination, resulted in space-time yields of up to 12.7 g L-1 h(-1). The method achieved 98.3% substrate conversion and 99.2% syn de within 6 h, marking the highest reported levels to date. The above findings will contribute to the industrial production of beta-hydroxy-alpha-amino acids, offer insights into the mechanism of Loop regions regulating the catalytic function of LTTAs, and provide ideas for engineering other enzymes. |
| WOS关键词 | SERINE HYDROXYMETHYL TRANSFERASE ; ALPHA-AMINO ACID ; STREPTOCOCCUS-THERMOPHILUS ; ENANTIOSELECTIVE SYNTHESIS ; ASYMMETRIC-SYNTHESIS ; DIRECTED EVOLUTION ; ESCHERICHIA-COLI ; ALDOLASE ; SUBSTRATE ; BINDING |
| 资助项目 | National Key Research and Development Program of China[2023YFA0914500] ; National Science Foundation of China[32271487] ; National First-class Discipline Program of Light Industry Technology and Engineering[LITE2018-12] ; Program of Introducing Talents of Discipline to Universities[111-2-06] |
| WOS研究方向 | Chemistry |
| 语种 | 英语 |
| WOS记录号 | WOS:001255663200001 |
| 出版者 | AMER CHEMICAL SOC |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/312056]  |
| 专题 | 新药研究国家重点实验室 |
| 通讯作者 | Zhang, Rongzhen |
| 作者单位 | 1.Johns Hopkins Univ, Sch Med, Solomon H Snyder Dept Neurosci, Baltimore, MD 21205 USA 2.Chinese Acad Sci, Shanghai Inst Mat Med, Drug Discovery & Design Ctr, State Key Lab Drug Res, 555 Zuchongzhi Rd, Shanghai 201203, Peoples R China 3.Jiangnan Univ, Key Lab Ind Biotechnol, Minist Educ, Wuxi 214122, Peoples R China 4.Jiangnan Univ, Sch Biotechnol, Wuxi 214122, Peoples R China |
| 推荐引用方式 GB/T 7714 |
Xi, Zhiwen,Rao, Jingxin,Zhang, Xinyi,et al. Deciphering the Key Loop: Enhancing |
| APA |
Xi, Zhiwen.,Rao, Jingxin.,Zhang, Xinyi.,Liu, Zhiyong.,Zheng, Mingyue.,...&Zhang, Rongzhen.(2024).Deciphering the Key Loop: Enhancing |
| MLA |
Xi, Zhiwen,et al."Deciphering the Key Loop: Enhancing |
入库方式： OAI收割
来源：上海药物研究所
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