Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Abstract

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP-free intermediate towards the final metabolite. However, the timing of ACP off-loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone-2-carboxylic acid-N-acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)-DNPA, and the product of ActIV reaction was characterized as an ACP-free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC-TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.

Total Synthesis of the Nonribosomal Peptide Surugamide B and Identification of a New Offloading Cyclase Family

“…[3] It is also different from the type II TE [14] and other known discrete offloading counterparts found in TE-less polyketide synthases (PKSs), such as SgcE for C-1027, [15] metallo-b-lactamase for fungal type IPKSs, [16] and zinc metallo-hydrolase for type II PKSs in actinomycete. [17] Instead, it is similar to penicillin-binding proteins (PBPs), [13] which catalyze transpeptidation/carboxypeptidation in bacterial cell wall synthesis through the formation of an acyl-enzyme intermediate,inwhich an activesite serine residue is transiently acylated. TheP BP family proteins share the characteristic catalytic tetrad, Ser-Lys-Tyr-His,w hich is also found in the SurE sequence ( Figure S14).…”

Unveiling Two Consecutive Hydroxylations: Mechanisms of Aromatic Hydroxylations Catalyzed by Flavin‐Dependent Monooxygenases for the Biosynthesis of Actinorhodin and Related Antibiotics

“…Position number is based on the polyketide (C16) biosynthetic origin. A common bicyclic intermediate undergoes stereospecific keto reductions at C‐3/C‐15, leading either to 2 or to 12 . Two hydroxylations at C‐6/C‐8 or a single hydroxylation at C‐6 are involved in ACT/GRA and MED biosynthesis, respectively .…”

“…The fact that 2 is converted into 8 through the action of ActVA‐5/ActVB supports our previous proposal that 11 would be a substrate for dimerization through regiospecific aryl coupling, the last step in ACT biosynthesis. Recently, we established an in vitro reconstitution system using recombinant ACT biosynthetic enzymes to produce 3 . In S. coelicolor , an enoylreductase encoded by act VI‐ORF2 converts 3 stereospecifically into 2 (Scheme ) .…”

Total Synthesis of the Nonribosomal Peptide Surugamide B and Identification of a New Offloading Cyclase Family

“…28 kDa) . It is also different from the type II TE and other known discrete offloading counterparts found in TE‐less polyketide synthases (PKSs), such as SgcE for C‐1027, metallo‐β‐lactamase for fungal type I PKSs, and zinc metallo‐hydrolase for type II PKSs in actinomycete . Instead, it is similar to penicillin‐binding proteins (PBPs), which catalyze transpeptidation/carboxypeptidation in bacterial cell wall synthesis through the formation of an acyl‐enzyme intermediate, in which an active‐site serine residue is transiently acylated.…”