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

Abstract

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a ␤-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.Modular polyketide synthases (PKSs) 5 and non-ribosomal peptide synthetases (NRPSs) are large multienzymes that catalyze the production of a wide range of biologically active compounds currently used as therapies for human diseases (1). Most PKSs and NRPSs are organized as assembly lines in which specific enzymatic domains catalyze elongation or modification of specific pathway intermediates. One key difference between these two types of assembly lines is that PKSs use acyl thioesters as substrates, whereas NRPSs use amino acids. Both PKS and NRPS pathway intermediates are tethered, via a thioester bond, to the phosphopantetheine (Ppant) arms of acyl/ peptidyl carrier protein (ACP/PCP) domains throughout chain assembly. Thioester cleavage, most often catalyzed by a terminating or type I thioesterase (TE I), releases the fully assembled polyketide/peptide from the carrier domain.Prodiginine biosynthesis is directed by the red cluster in Streptomyces coelicolor and involves hybrid NRPS/PKS multienzymes employing an ␣-oxoamine synthase (OAS) in a highly unusual chain release mechanism to assemble undecylprodiginine (1) and its carbocyclic derivative streptorubin B (2) (Fig. 1A) (2-5). The therapeutic potential of prodiginines was demonstrated in prior studies in which analogues of undecylprod...

Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

“…However, there might well be a (yet-unknown) transferase which directly transfers long-chain acyl moieties from ACP to CoA. Streptomyces TEs have not been characterized in the context of storage lipid synthesis yet, but the involvement of several TEs in PKS was studied (67)(68)(69). Type II TEs are dislocated from the multimodular PKS machinery and remove acyl residues from the extension modules.…”

“…Type II TEs are dislocated from the multimodular PKS machinery and remove acyl residues from the extension modules. However, some were reported to be selective for PKS-related ACP versions and might therefore not function with FAS II-derived acyl-ACPs (67,68). The genome of G25 encodes putative TEs with approximately 30 to 50% amino acid identity to the well-characterized type II TE (TesB) from E. coli (70) (STSP_45110 and STSP_26350 [ Table 3]).…”

Bioprospecting Anticancer Compounds from the Marine-Derived Actinobacteria Actinomadura sp. Collected at the Saint Peter and Saint Paul Archipelago (Brazil)

“…RedJ biochemical characterization corroborates the proposed activity by proving this thioesterase strong selectivity for long and saturated acyl chains that are linked to prodiginine specific RedQ ACP in detriment to substrates attached to FabC, the fatty acid synthase ACP of streptomycete. 31 However, no differential kinetics was observed for RedJ when cleaving dodecanoyl or decanoylAcpP substrates, indicating that this thioesterase may also have editing activities related to deacetylation of ACP/PCP (peptidyl carrier protein) domains missacetylated by 4'-phosphopantetheinyl transferase (PPTase) RedU. 25,31 In this case, RedJ would not have the fine capability to discriminate between the two long chain substrates, and RedL_A would be the key component for selecting decanoyl substrate on the prodiginine biosynthetic pathway of Actinomadura spp.…”

“…25,31 In this case, RedJ would not have the fine capability to discriminate between the two long chain substrates, and RedL_A would be the key component for selecting decanoyl substrate on the prodiginine biosynthetic pathway of Actinomadura spp.…”

Expression, crystallization and preliminary crystallographic data analysis of PigI, a putativeL-prolyl-AMP ligase from the prodigiosin synthetic pathway inSerratia

“…The l-prolyl-S-PigG is then desaturated to pyrrolyl-2-carboxyl-S-PigG by PigA, a 42 kDa flavoprotein desaturase (Garneau-Tsodikova et al, 2006). Several crystallographic studies on the gene products in the prodigiosin biosynthetic gene cluster have been attempted, but none of them concerns the initiation of the MBC synthetic pathway (Whicher et al, 2011;Liu et al, 2012;Cho et al, 2008). To elucidate the mechanism of the first step of MBC biosynthesis, we cloned and expressed the PigI gene from the previously isolated Serratia sp.…”