The product, 10-deoxymethynolide, contains a 12-membered ring and is an intermediate in the biosynthesis of methymycin in the bacterium Streptomyces venezuelae. The enzyme also produces narbonolide (see EC 2.3.1.240, narbonolide synthase). The enzyme has 29 active sites arranged in four polypeptides (pikAI - pikAIV) with a loading domain, six extension modules and a terminal thioesterase domain. Each extension module contains a ketosynthase (KS), keto reductase (KR), an acyltransferase (AT) and an acyl-carrier protein (ACP). Not all active sites are used in the biosynthesis.
electron cryo-microscopy (cryo-EM) structures of a full-length polyketide synthase module in three key biochemical states of its catalytic cycle reveals the dynamics of the intramodule acyl carrier protein for sequential binding to catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the polyketide synthase pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the acyl carrier protein-bound polyketide chain elongation intermediate
The product, 10-deoxymethynolide, contains a 12-membered ring and is an intermediate in the biosynthesis of methymycin in the bacterium Streptomyces venezuelae. The enzyme also produces narbonolide (see EC 2.3.1.240, narbonolide synthase). The enzyme has 29 active sites arranged in four polypeptides (pikAI - pikAIV) with a loading domain, six extension modules and a terminal thioesterase domain. Each extension module contains a ketosynthase (KS), keto reductase (KR), an acyltransferase (AT) and an acyl-carrier protein (ACP). Not all active sites are used in the biosynthesis.
pikromycin modules PikAIII and PikAIV generate the 12-membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. PikAIV adopts an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization
the engineered polyketide synthase PikAIII-TE(thioesterase domain) fusion protein accepts and processes the pentaketide to produce 10-deoxymethynolide as the sole product. The polyketide synthase PikAIII/polyketide synthase PikAIV complex processes the pentaketide to produce 10-deoxymethynolide and narbonolide. After incubation of the hexaketide with polyketide synthase PikAIII-TE(thioesterase domain) fusion protein in both the presence and absence of NADPH, no product formation is observed
the enzyme also produces narbonolide (see EC 2.3.1.240, narbonolide synthase). The enzyme has 29 active sites arranged in four polypeptides (pikAI - pikAIV) with a loading domain, six extension modules and a terminal thioesterase domain. Each extension module contains a ketosynthase (KS), keto reductase (KR), an acyltransferase (AT) and an acyl-carrier protein (ACP). Not all active sites are used in the biosynthesis
despite clear similarities in biochemistry and underlying module organization the picromycin synthase modules 5+TE(thioesterase) and 6+TE(thioesterase) show clear differences in substrate specificity and tolerance
by modification of the type of hexaketide ester employed, product formation may be controled with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring
both PikAIV(DELTAAT) and PikAIV(DELTAACP) display similar apparent kcat values for 10-deoxymethynolide production, which are approximately 2fold lower than the calculated kcat from wild-type PikAIII and PikAIV. Likewise, both PikAIV mutants display similar apparent KM values that are also reduced 2fold when compared to the KM from PikAIII and PikAIV. The equivalent decrease in the apparent kinetic parameters for both mutants results in a specificity constant (kcat/KM) that is comparable to wild-type, further emphasizing the non-critical role of these catalytic domains in 10-deoxymethynolide production. Both PikAIII(DELTADock) (partial deletions of PikAIII C-terminal domain) and PikAIV(DELTADock) (partial deletion of PikAIV N-terminal docking domains) display apparent kcat values (0.044/min and 0.053/min), which are significantly decrease relative to the kcat observed with wild-type PikAIII and PikAIV. The apparent KM values for PikAIII(DELTADock) and PikAIV (DELTADock) decrease 35 fold compared to wild-type, resulting in specificity constants (kcat/KM) that are 14fold and 8fold lower, respectively. Combining PikAIII(DELTADock) and PikAIV(DELTADock) together do not result in an additional rate decrease. The kinetic parameters are comparable to those obtained when either truncated monomodule is paired with its wild-type partner
Q9ZGI5: type I polyketide synthase PikAI, Q9ZGI4: type I polyketide synthase PikAII, Q9ZGI3: type I polyketide synthase PikAIII, Q9ZGI2: type I polyketide synthase PikAIV, Q9ZGI1: thioesterase II PikAV. Multifunctional, modular enzyme
in modular type I polyketide synthases the presence of the three processing domains, i.e. ketoreductase (KR), dehydratase (DH), and enoylreductase (ER), are varied in each module, leading to a fully reduced, partially reduced, or unreduced segment on the polyketide chain. Dehydratase PikDH2 converts D-alcohol substrates to trans-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. The enzyme activity is robust, and PikDH2 can be used for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 shows remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the beta-, gamma-, or delta-position
pikromyin polyketide synthase is comprised of a loading module and six extension modules. PikAI is a multimodular component of this pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers
pikromyin polyketide synthase is comprised of a loading module and six extension modules. PikAI is a multimodular component of this pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers
multimodular separation can lead to only a modest decrease in the overall production of the final polyketide production. PikAI is a multimodular component of the pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the Streptomyces venezuelae mutant are 50% of that of a Streptomyces venezuelae strain expressing the native trimodular PikAI. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain is also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate
multimodular separation can lead to only a modest decrease in the overall production of the final polyketide production. PikAI is a multimodular component of the pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the Streptomyces venezuelae mutant are 50% of that of a Streptomyces venezuelae strain expressing the native trimodular PikAI. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain is also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate
PikAI is a multimodular component of the pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin polyketide synthase are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the Streptomyces venezuelae mutant are 50% of that of a Streptomyces venezuelae strain expressing the native trimodular PikAI. This observation provides evidence that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain is also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of docking domains to manipulate biosynthetic processes catalyzed by modular polyketide synthases and the quest to generate novel polyketide products
PikAI is a multimodular component of the pikromyin polyketide synthase and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin polyketide synthase are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the Streptomyces venezuelae mutant are 50% of that of a Streptomyces venezuelae strain expressing the native trimodular PikAI. This observation provides evidence that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain is also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of docking domains to manipulate biosynthetic processes catalyzed by modular polyketide synthases and the quest to generate novel polyketide products
the picromycin/methymycin PKS genes (pikAI, PikAII, PikAIII, PikAIV and pikAV) are functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide
substrate engineering approaches to control the catalytic cycle of a full polykeitde synthase module harboring multiple domains. Using alternatively activated native hexaketide substrates, product formation may be controled with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring
versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways are hybridized in Saccharomyces cerevisiae to generate hybrid libraries. Streptomyces venezuelae strains that expressed active chimeric enzymes with new functionality are capable of producing engineered macrolactones
Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase
Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains
ChemBioChem
10
1537-1543
2009
Streptomyces venezuelae (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1), Streptomyces venezuelae, Streptomyces venezuelae ATCC 15439 (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1)
Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase
J. Am. Chem. Soc.
125
5671-5676
2003
Streptomyces venezuelae (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1), Streptomyces venezuelae ATCC 15439 (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1)
Structural rearrangements of a polyketide synthase module during its catalytic cycle
Nature
510
560-564
2014
Streptomyces venezuelae (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1), Streptomyces venezuelae, Streptomyces venezuelae ATCC 15439 (Q9ZGI5 and Q9ZGI4 and Q9ZGI3 and Q9ZGI2 and Q9ZGI1)