Efficient Synthesis of Aromatic Polyketides <i>in Vitro</i> by the Actinorhodin Polyketide Synthase

Carreras, Christopher W.; Pieper, Rembert; Khosla, Chaitan

doi:10.1021/ja960479o

Cited by 38 publications

(50 citation statements)

References 22 publications

(39 reference statements)

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“…More recently, similar experiments were reported using a system in which genes for the actinorhodin (act) PKS were expressed in a recombinant strain of S. coelicolor from which the entire act gene cluster had been deleted [107]. This work demonstrated the efficient conversion of acetyl and malonyl CoA into two aromatic polyketide products, SEK 4 and SEK4b, by cell-free preparations of the act minimal PKS (Fig.…”

Section: Bacterial Aromatic Polyketide Synthasessupporting

confidence: 66%

“…In large measure this has been due to the absence of fully active cell-free systems for the study of these enzymes. Recently however, the situation has changed [ 106,107], and these in vitro systems are expected to facilitate the purification of the responsible proteins, followed by their kinetic, physicochemical, and ultimately structural characterization.…”

Section: Bacterial Aromatic Polyketide Synthasesmentioning

confidence: 99%

See 1 more Smart Citation

The chemistry and biology of fatty acid, polyketide, and nonribosomal peptide biosynthesis

Carreras

Pieper

Khosla

1997

Topics in Current Chemistry

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Polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases are a structurally and mechanistically related class of enzymes that catalyze the synthesis of biopolymers in the absence of a nucleic acid or other template. These enzymes utilize the common mechanistic feature of activating monomers for condensation via covalently-bound thioesters of phosphopantetheine prosthetic groups. The information for the sequence and length of the resulting polymer appears to be encoded entirely within the responsible proteins.Polyketide and fatty acid biosyntheses begin with condensation of the coenzyme A thioester of a short-chain carboxylic acid "starter unit" such as acetate or propionate with the coenzyme A thioester of a dicarboxylic acid "extender unit" such as malonate or methyl malonate. The driving force for the condensation is provided by the decarboxylation of the extender unit. In the case of fatty acid synthesis, the resulting fl-carbonyl is completely reduced to a methylene; however, during the synthesis of complex polyketides, the fl-carbonyl may be left untouched or variably reduced to alcohol, olefinic, or methylene functionalities depending on the position that the extender unit will occupy in the final product. This cycle is repeated, and the number of elongation cycles is a characteristic of the enzyme catalyst. In polyketide biosynthesis, the full-length polyketide chain cyclizes in a specific manner, and is tailored by the action of additional enzymes in the pathway.Several architectural paradigms are known for polyketide and fatty acid synthases. While the bacterial enzymes are composed of several monofunctional polypeptides which are used during each cycle of chain elongation, fatty acid and polyketide synthases in higher organisms are multifunctional proteins with an individual set of active sites dedicated to each cycle of condensation and ketoreduction. Peptide synthetases also exhibit a one-to-one correspondence between the enzyme sequence and the structure of the product. Together, these systems represent a unique mechanism for the synthesis of biopolymers in which the template and the catalyst are the same molecule.

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Section: Bacterial Aromatic Polyketide Synthasessupporting

confidence: 66%

Section: Bacterial Aromatic Polyketide Synthasesmentioning

confidence: 99%

The chemistry and biology of fatty acid, polyketide, and nonribosomal peptide biosynthesis

Carreras

Pieper

Khosla

1997

Topics in Current Chemistry

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“…Initial efforts toward these goals relied on metabolic characterization of genetically manipulated PKSs. More recently, cell-free systems have been developed to study polyketide synthesis in vitro (5,6,8). The actinorhodin (act) PKS from S. coelicolor has been an excellent model system for both in vivo and in vitro studies.…”

Section: Discussionmentioning

confidence: 99%

“…Repeated decarboxylative condensations occur between the ACP-bound nucleophilic extender units and the KSbound electrophilic growing chains, giving rise to a poly-␤-ketone backbone of a specified length (5). (The system is primed by a decarboxylated malonyl unit by a mechanism that remains to be elucidated, but presumably involves the decarboxylative activity of the KS (8).) In the case of the actinorhodin (act) minimal PKS, a 16-carbon backbone is generated, which subsequently undergoes cyclization to generate two principal products, SEK4 and SEK4b (Fig.…”

mentioning

confidence: 99%

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Dreier¹,

Shah²,

Khosla³

1999

Journal of Biological Chemistry

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Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA: ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-␤-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of proteinprotein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 ؎ 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [ 14 C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

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“…This variability could either arise due to differences in the substrate binding pockets of individual subunits, or it may reflect differential affinities for the minimal PKS subunits themselves. Given the availability of cell-free systems for polyketide biosynthesis (28,29), further in vitro analysis could shed light on this question.…”

Section: Discussionmentioning

confidence: 99%

Domain Analysis of the Molecular Recognition Features of Aromatic Polyketide Synthase Subunits

Zawada¹,

Khosla²

1997

Journal of Biological Chemistry

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Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/ CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.

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Efficient Synthesis of Aromatic Polyketides in Vitro by the Actinorhodin Polyketide Synthase

Cited by 38 publications

References 22 publications

The chemistry and biology of fatty acid, polyketide, and nonribosomal peptide biosynthesis

The chemistry and biology of fatty acid, polyketide, and nonribosomal peptide biosynthesis

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Domain Analysis of the Molecular Recognition Features of Aromatic Polyketide Synthase Subunits

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