5tic

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

Structural highlights 5tic is a 2 chain structure with sequence from Escherichia coli. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.65Å Ligands: Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function TESA_ECOLI Hydrolyzes only long chain acyl thioesters (C12-C18). Specificity similar to chymotrypsin. Publication Abstract from PubMed 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 (C6-C12) 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 C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 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 in vitro 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. Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids.,Grisewood MJ, Hernandez Lozada NJ, Thoden JB, Gifford NP, Mendez-Perez D, Schoenberger HA, Allan MF, Floy ME, Lai RY, Holden HM, Pfleger BF, Maranas CD ACS Catal. 2017 Jun 2;7(6):3837-3849. doi: 10.1021/acscatal.7b00408. Epub 2017, Apr 20. PMID:29375928 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Thioesterase 3D structures

spinqualitylabelsall models 5tic, resolution 1.65Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image