5TIC

X-ray structure of wild-type E. coli Acyl-CoA thioesterase I at pH 5

5TIC の概要

• エントリーDOI: 10.2210/pdb5tic/pdb
• 関連するPDBエントリー: 5TID 5TIE 5TIF
• 分子名称: Acyl-CoA thioesterase I, CHLORIDE ION (3 entities in total)
• 機能のキーワード: thioesterase, tesa, fatty acid, hydrolase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 41478.52

構造登録者

Thoden, J.B.,Holden, H.M.,Grisewood, M.J.,Hernandez Lozada, N.J.,Gifford, N.P.,Mendez-Perez, D.,Schoenberger, H.A.,Allan, M.F.,Pfleger, B.F.,Marines, C.D. (登録日: 2016-10-02, 公開日: 2017-04-26, 最終更新日: 2023-10-04)

主引用文献

Grisewood, M.J.,Hernandez Lozada, N.J.,Thoden, J.B.,Gifford, N.P.,Mendez-Perez, D.,Schoenberger, H.A.,Allan, M.F.,Floy, M.E.,Lai, R.Y.,Holden, H.M.,Pfleger, B.F.,Maranas, C.D.
Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids.
ACS Catal, 7:3837-3849, 2017

PubMed Abstract: Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C-C) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C-C oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in We used the IPRO algorithm to design thioesterase variants with enhanced C- or C-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C) and twenty-seven thioesterases with enhanced production of octanoic acid (C). The top variants reached up to 49% C and 50% C while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of 'TesA and the best mutant were confirmed using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of 'TesA variants suggest that hydrophobic forces govern 'TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.

PubMed: 29375928
DOI: 10.1021/acscatal.7b00408

実験手法

X-RAY DIFFRACTION (1.65 Å)