PDBsum entry 5tic

Hydrolase

PDB id: 5tic

Contents

Protein chains

177 a.a.

Metals

_CL

Waters ×346

PDB id:

5tic

Name:

Hydrolase

Title:

X-ray structure of wild-type e. Coli acyl-coa thioesterase i at ph 5

Structure:

Acyl-coa thioesterase i. Chain: a, b. Fragment: unp residues 28-208. Synonym: lecithinase b,lysophospholipase l1,protease i. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: tesa, apea, pldc, b0494, jw0483. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.65Å R-factor: 0.188 R-free: 0.222

Authors:

J.B.Thoden,H.M.Holden,M.J.Grisewood,N.J.Hernandez Lozada,N.P.Gifford, D.Mendez-Perez,H.A.Schoenberger,M.F.Allan,B.F.Pfleger,C.D.Marines

Key ref:

M.J.Grisewood et al. (2017). Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids. ACS Catal, 7, 3837-3849. PubMed id: 29375928

Date:

02-Oct-16 Release date: 26-Apr-17

Protein chains

P0ADA1  (TESA_ECOLI) -  Thioesterase 1/protease 1/lysophospholipase L1 from Escherichia coli (strain K12)

Seq: 208 a.a.

Struc: 177 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: E.C.3.1.1.2 - arylesterase.
• Reaction: a phenyl acetate + H2O = a phenol + acetate + H⁺
• Enzyme class 3: E.C.3.1.1.5 - lysophospholipase.
• Reaction: a 1-acyl-sn-glycero-3-phosphocholine + H2O = sn-glycerol 3-phosphocholine + a fatty acid + H⁺
• Enzyme class 4: E.C.3.1.2.14 - oleoyl-[acyl-carrier-protein] hydrolase.
• Reaction: (9Z)-octadecenoyl-[ACP] + H2O = (9Z)-octadecenoate + holo-[ACP] + H⁺
• Enzyme class 5: E.C.3.1.2.2 - palmitoyl-CoA hydrolase.
• Reaction: hexadecanoyl-CoA + H2O = hexadecanoate + CoA + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

ACS Catal 7:3837-3849 (2017)

PubMed id: 29375928

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids.

M.J.Grisewood, N.J.Hernandez Lozada, J.B.Thoden, N.P.Gifford, D.Mendez-Perez, H.A.Schoenberger, M.F.Allan, M.E.Floy, R.Y.Lai, H.M.Holden, B.F.Pfleger, C.D.Maranas.

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 (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-C₁₀oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase inEscherichia 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% C₁₂and 50% C₈while 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 confirmedin vitrousing 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.