Biochemical Evidence for an Editing Role of Thioesterase II in the Biosynthesis of the Polyketide Pikromycin

Abstract

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6- Polyketides are a large and structurally diverse class of natural products that possess a wide range of biological activities (1). These compounds are used throughout medicinal and agricultural fields as antimicrobials, immunosuppressants, antiparasitics, and anticancer agents. Despite their structural diversity, polyketides are assembled by a common mechanism of decarboxylative condensations of simple malonate derivatives by polyketide synthases (PKSs) 1 in a manner very similar to fatty acid biosynthesis (2, 3). Type I PKSs are a family of PKSs that are analogous to vertebrate fatty acid synthase that catalyze the biosynthesis of the polyketide moieties of various secondary metabolites in Streptomyces (4 -6). They are gigantic multifunctional modular proteins. Each module is responsible for one cycle of polyketide chain elongation and contains a set of discrete catalytic domains of ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP). Ketoreductase, dehydratase (DH), and enoyl reductase domains may also be present, allowing structural variation in the level of processing of the ␤-ketoacyl chain (4, 6). The fully extended polyketide chain bound to the PKS as an acyl-ACP thioester is often released and cyclized by a thioesterase domain (TEI) covalently linked to the last extending module of the PKS (7).In many cases, additional genes encoding a TE have been found within a polyketide biosynthetic gene cluster, for example, the tylosin PKS of Streptomyces fradiae (8), pikromycin PKS of Streptomyces venezuelae (5), rifamycin PKS of Amycolatopsis mediterranei (9), and the erythromycin PKS (DEBS) of Saccharopolyspora erythraea (10). These genes encoding a discrete protein were named as TEII to differentiate from the chain releasing TEI domains in modular polyketide synthases. Discrete TEII enzymes are also associated with bacterial nonribosomal peptide synthases, responsible for the production of macrocyclic peptide compounds (11), and animal fatty acid synthases (12). Sequence analysis has revealed that these thioesterases are probably structurally and evolutionarily related (13). They have a common...

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

“…For example, RifR hydrolyzes both long-chain and carboxylated acyl-CoAs and has at most 30-fold selectivity for ACP-over CoA-thioesters. The low activity and low substrate selectivity of RifR are consistent with its proposed editing function, which requires the hydrolysis of a range of aberrant intermediates that stall natural product biosynthesis (13,16,17). The stronger substrate selectivity of RedJ suggests that editing is not its primary function.…”

“…Such nonproductive intermediates may arise from premature decarboxylation by a KS domain (16,17) or incorrect priming of carrier protein domains (15,18). Consistent with the proposed editing function, previously characterized TE IIs exhibit low substrate selectivity, hydrolyze a range of substrates from a range of ACPs, and have low activity (13,16,17).…”

“…Based on sequence similarity and genetic studies, previous reports have assigned RedJ an editing function similar to TE IIs from other natural product biosynthetic pathways (11,14). TE IIs are thought to relieve stalled assembly lines by removing non-productive intermediates from carrier protein domains (13,(15)(16)(17)(18). Such nonproductive intermediates may arise from premature decarboxylation by a KS domain (16,17) or incorrect priming of carrier protein domains (15,18).…”

Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

“…TEIIs from different pathways have differing specificities, but general trends include a preference for decarboxylated acyl units over carboxylated acyl units (5,6,27), substrates linked to a carrier domain over substrates linked to CoA or the phosphopantetheine mimic N-acetylcysteamine (7,28), and single amino acids over di-or tri-peptides (6,7). TEIIs are able to hydrolyze substrates attached to carrier domains from their native pathway as well as other pathways (6,20,28).…”

Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis

“…Disruption of the corresponding TEII genes in the producer strains inhibited product formation by 80-90% [12][13][14]. Recently, biochemical studies on TEIIs in polyketide synthesis suggested a role in the removal of short acyl chains originating from aberrant decarboxylation of chain extender units from the thiol moiety of the 4¢-Ppant cofactors of acyl carrier proteins [15,16]. In NRPSs there is no such decarboxylation process during product synthesis.…”