Expression, Site-Directed Mutagenesis, and Steady State Kinetic Analysis of the Terminal Thioesterase Domain of the Methymycin/Picromycin Polyketide Synthase

Abstract

The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.

Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate

“…5A). The molecular mass of the main compound (compound a) was 30,718.48 Ϯ 0.44 Da (molecule [MϩH] ϩ ), which is in agreement with the expected molecular mass of recombinant ScoT protein lacking the N-terminal methionine (30,717.98 Da). The second peak (compound b) represented the same protein with a gluconic acid residue attached to its N terminus.…”

Enhancement and Selective Production of Phoslactomycin B, a Protein Phosphatase IIa Inhibitor, through Identification and Engineering of the Corresponding Biosynthetic Gene Cluster

“…Each of the remaining components, PLM4 -PLM7, contains a single module. PLM7 has a thioesterase domain, consistent with a role in catalyzing the last extension step and subsequent release of the polyketide chain by formation of the ␣,␤-unsaturated ␦-lactone (formation of a six-member lactone is unusual, as most thioesterase domains studied to date catalyze formation of large 12-14 macrolactone structures under natural conditions) (37,38). Two of the PKS polypeptides PLM4 and PLM6, contain the same catalytic domains, which precludes unambiguous assignment in this process.…”

Understanding Substrate Specificity of Polyketide Synthase Modules by Generating Hybrid Multimodular Synthases

“…Expression plasmids pKW127 (picM3ϩpicTE) were constructed by replacing the BsaBI-EcoRI fragments containing eryM3ϩeryTE in pRSG34 with the corresponding modules fused to the picTE domain. Again (23,24), both polypeptides retained the N-terminal linker region of eryM3. Expression plasmid pKW198 (picM6ϩpicTE) was constructed by replacing the BsaBI-HindIII fragment containing eryM3ϩeryTE in pRSG34 with the fragment containing picM6ϩpicTE.…”