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

Abstract

Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an ␣/␤ hydrolase with Ser90 and His224 in its active site.

An Unusual Thioesterase Promotes Isochromanone Ring Formation in Ajudazol Biosynthesis

“…This contrasts with the TE domain of the lasalocid PKS, which clades with typical type I TEs. Based on the literature precedent, [17,19] AjuTE would be expected to show a preference in the deacylation reaction for short acyl chains such as acetate and propionate, consistent with its role in removing aberrant acyl units from the carrier-protein domains. As TEIIs are encoded by discrete genes, the in silico analysis also raised the possibility that the ajuTE gene had once been a stand-alone element, but had become fused genetically to the end of gene ajuH.…”

“…acylation of the acyl-enzyme intermediate has been shown to be the rate-determining step in turnover of the p-NP esters by both type I and II TEs. [17,19,35] As this is the stage at which the isochromanone ring would be formed in the normal pathway, it was of particular interest to characterize TE substrate specificity during this half of the reaction. One disadvantage of using these esters, however, is that substrate-saturating conditions cannot be attained due to their poor aqueous solubility, and therefore it is not possible to determine apparent k cat and K M values.…”

“…Nonetheless, meaningful information has been obtained by comparing the apparent specificity constants (k cat / K M ) calculated from the initial rates of reaction at low concentrations of various substrates. [17,19] All TEs were assayed in 96-well plate format with p-NP esters 3-6 as substrates (Table 1). Based on preliminary experiments to determine the limiting concentration of each substrate, the compounds were evaluated at six concentrations within the solubility range (0.05-5 mm, depending on the substrate).…”

Biochemical and Genetic Insights into Asukamycin Biosynthesis

“…As the yields of the polyketide and polypeptide products were often considerably impaired in the knock-out mutants, type II thioesterases were proposed to restore the acyl or peptidyl carrier protein function by releasing undesirable intermediates, resulting from a priming or processing error (51)(52)(53)(54). The asuC15 mutant produced a normal amount of asukamycin A1, yet the yield of the congeners A2-A7 was significantly reduced (Fig.…”

Coordinative Modulation of Chlorothricin Biosynthesis by Binding of the Glycosylated Intermediates and End Product to a Responsive Regulator ChlF1

“…BLAST analysis indicated that ChlK is a type II thioesterase, which probably removes aberrantly loaded substrates from the polyketide chain to promote the specific loading efficiency (28,29). Both HPLC analysis and bioassays showed that the CHL production in ⌬chlK was distinctly lower than that in wild type and was almost fully restored in the complementary strain (⌬chlK/pLY108).…”