Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis.
ABSTRACT: Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS) pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold.
Project description:Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues exist within the "GNAT" (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl, or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl ?-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.
Project description:The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (?GNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ?GNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.
Project description:General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections.
Project description:Enzymes belonging to the GNAT superfamily are widely distributed in nature where they play key roles in the transfer of acyl groups from acyl-CoAs to primary amine acceptors. The amine acceptors run the gamut from histones to aminoglycoside antibiotics to small molecules such as serotonin. Whereas those family members that function on histones have been extensively studied, the GNAT enzymes that employ nucleotide-linked sugars as their substrates have not been well characterized. Indeed, though the structures of two of these "amino sugar" GNAT enzymes have been determined within the past 10 years, details concerning their active site architectures have been limited because of a lack of bound nucleotide-linked sugar substrates. Here we describe a combined structural and biochemical analysis of FdhC from Acinetobacter nosocomialis O2. On the basis of bioinformatics, it was postulated that FdhC catalyzes the transfer of a 3-hydroxybutanoyl group from 3-hydroxylbutanoyl-CoA to dTDP-3-amino-3,6-dideoxy-d-galactose, to yield an unusual sugar that is ultimately incorporated into the surface polysaccharides of the bacterium. We present data confirming this activity. In addition, the structures of two ternary complexes of FdhC, in the presence of CoA and either 3-hydroxybutanoylamino-3,6-dideoxy-d-galactose or 3-hydroxybutanoylamino-3,6-dideoxy-d-glucose, were solved by X-ray crystallographic analyses to high resolution. Kinetic parameters were determined, and activity assays demonstrated that FdhC can also utilize acetyl-CoA, 3-methylcrotonyl-CoA, or hexanoyl-CoA as acyl donors, albeit at reduced rates. Site-directed mutagenesis experiments were conducted to probe the catalytic mechanism of FdhC. Taken together, the data presented herein provide significantly new molecular insight into those GNAT superfamily members that function on nucleotide-linked amino sugars.
Project description:Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, ?-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and ?-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the ?-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.
Project description:Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP(I)-ACP(II)-ACP(III)) within the CurA PKS module. We have determined the structure of the isolated holo-ACP(I) and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP(I), which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP(I) and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP(I)-ACP(II)-ACP(III) tridomain, our data provide insight into the specificity of the chlorination reaction.
Project description:Gcn5-related N-acetyltransferases (GNATs) are found in all kingdoms of life and catalyze important acyl transfer reactions in diverse cellular processes. While many 3D structures of GNATs have been determined, most do not contain acceptor substrates in their active sites. To expand upon existing crystallographic strategies for improving acceptor-bound GNAT structures, we synthesized peptide substrate analogs and reacted them with CoA in PA4794 protein crystals. We found two separate mechanisms for bisubstrate formation: (a) a novel X-ray induced radical-mediated alkylation of CoA with an alkene peptide and (b) direct alkylation of CoA with a halogenated peptide. Our approach is widely applicable across the GNAT superfamily and can be used to improve the success rate of obtaining liganded structures of other acyltransferases.
Project description:Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
Project description:The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.
Project description:GCN5-type N-acetyltransferases (GNATs) are enzymes that catalyse the transfer of the acetyl group from acetyl-CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side-chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT-catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in Streptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify SlPatA, a GNAT in Streptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl-CoA synthetase (SlAacS). The latter has homologues in all domains of life. In vitro and in vivo evidence show that SlAacS is a bona fide acetoacetyl-CoA synthetase. SlPatA acetylates SlAacS more efficiently than it does acetyl-CoA synthetase, an enzyme known to be under acetylation control. SlPatA acetylates SlAacS at the active-site residue Lys617 and acetylation inactivates SlAacS. Acetylated SlAacS was deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl-CoA synthetase activity in all domains of life.