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

Abstract

Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.

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

“…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).…”

“…Crystal and NMR solution structures of chain-terminating TE Is (19 -24) and TE IIs (13,25) from PKS and NRPS pathways demonstrate structural variability among TEs. All PKS and NRPS TEs adopt an ␣/␤-hydrolase fold and contain a serinehistidine-aspartate catalytic triad.…”

“…One candidate thioesterase is RedJ, which has high sequence similarity to editing, or type II, thioesterases (TE II), such as RifR (38% sequence identity) from the rifamycin pathway in Amycolatopsis mediterranei (11,13). 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).…”

“…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).…”

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

“…From comparison of the two B. fulva BGCs and the other BGCs identified above, four highly conserved genes were chosen for heterologous expression experiments in Aspergillus oryzae NSAR1:19, 20 the hrPKS bfpks1 ; the citrate‐synthase‐like gene bfL2 ; the methylcitrate dehydratase bfL3 ; and the hydrolase 341 bfL1 , which encodes a protein homologous to Type II thiolesterases such as RifR,21 which are involved in the release of abberant ACP‐bound polyketide intermediates during modular polyketide biosynthesis, as well as to LovG, which releases the lovastatin nonaketide from its PKS 22…”