RESUMO
Polyketide synthases (PKSs) are molecular assembly lines that condense basic chemical building blocks for the production of structurally diverse polyketides. Many PKS biosynthetic gene clusters contain a gene encoding for a type II thioesterase (TEII). It is believed that TEIIs exert a proofreading function and restore or increase the productivity of PKSs by removing aberrant modifications on the acyl-carrier proteins (ACPs) of the PKS assembly line. Yet biochemical evidence is still sparse. Here, we investigated the function of PnG, the TEII of the phoslactomycin PKS (Pn PKS), in the context of its cognate assembly line in vitro. Biochemical analysis revealed that PnG preferentially hydrolyzes alkyl-ACPs over (alkyl)malonyl-ACPs by up to three orders of magnitude, supporting a proofreading role of the enzyme. We further demonstrate that PnG increases the in vitro production of different native and non-native tetra-, penta-, and hexaketide derivatives of phoslactomycin by more than one order of magnitude and show that these effects are caused by the initial clearing of the Pn PKS, as well as proofreading of the active assembly line. Finally, we demonstrate that PnG is able to release intermediate but notably also terminal polyketides from the Pn PKS. This allows PnG to functionally replace and overcome the terminal TEI activity of chimeric in vitro Pn PKS systems, as showcased with a phoslactomycin hexaketide system. Altogether, our experiments provide detailed insights into the molecular mechanisms and the multiple functions of PnG in its native context, as well as their potential use in future applications.
Assuntos
Policetídeo Sintases , Policetídeos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Proteína de Transporte de AcilaRESUMO
Polyketide synthases (PKSs) use simple extender units to synthesize complex natural products. A fundamental question is how different extender units are site-specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS, which incorporates malonyl- and ethylmalonyl-CoA, as an inâ vitro model to study substrate specificity. We combined up to six Pn PKS modules with different termination sites for the controlled release of tetra-, penta- and hexaketides, and challenged these systems with up to seven different extender units in competitive assays to test for the specificity of Pn modules. While malonyl-CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl-CoA module PnC tolerates different α-substituted derivatives, but discriminates against malonyl-CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, thus explaining site-specific selectivity and promiscuity in the natural context of Pn PKS.
Assuntos
Policetídeo Sintases/metabolismo , Cinética , Lactonas/metabolismo , Compostos Organofosforados/metabolismo , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.
RESUMO
The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to change their chemical structure. Efforts to change extender unit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units. Here, we present a chemo-biosynthetic strategy that employs biocatalytic proofreading and allows access to a large variety of extender units. First, saturated acids are chemically coupled to free coenzyme A (CoA). The corresponding acyl-CoAs are then converted to alkylmalonyl-CoAs in a "one-pot" reaction through the combined action of an acyl-CoA oxidase and enoyl-CoA carboxylase/reductase. We synthesized six different extender units and used them in in vitro competition screens to investigate active site residues conferring extender unit selectivity. Our results show the importance of an uncharacterized glutamine in extender unit selectivity and open the possibility for comprehensive studies on extender incorporation in PKSs.