Structural analysis of protein–protein interactions in type I polyketide synthases

Xu, Wei; Qiao, Kangjian; Tang, Yi

doi:10.3109/10409238.2012.745476

Cited by 37 publications

(34 citation statements)

References 165 publications

(264 reference statements)

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“…Accordingly, it would be quite promising to develop a PKS system for obtaining hydrocarbon products with specific chain lengths, oxidation modifications, and branched-chain structures via rational design of the PKS and TE pair. However, carrying out a proof-of-principle study to demonstrate this may be difficult, for it is quite challenging to implement de novo designs or even to use the megaenzyme Type I PKS, due to the complexity of its intermodular and intramodular protein-protein interactions (Khosla et al, 2007;Xu et al, 2013). Over the past two decades, reprogramming modular Type I PKS has attracted considerable attention, and many ingenious strategies been yielded progress (Cane, 2010).…”

Section: Discussionmentioning

confidence: 98%

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Liu

Cheng

et al. 2015

Metabolic Engineering

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Section: Discussionmentioning

confidence: 98%

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Liu

Cheng

et al. 2015

Metabolic Engineering

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“…The functions of the KS domains from the PUFA synthase were further reaffirmed by site-directed mutagenesis and overexpression. The cysteines at the predicted active sites of these domains are highly conserved in discrete ketoacylsynthase enzymes and KS domains of fatty acid synthase and polyketide synthase in animals, plants, and microbes (14,32,(34)(35)(36)(37)(38). The thiolate form is believed to serve as an "nucleophilic elbow" necessary for the nucleophilic attack in the transacylation reaction (37).…”

Section: Discussionmentioning

confidence: 99%

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

Xie

Meesapyodsuk

Qiu

2017

Appl Environ Microbiol

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sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both and mutants. Overexpression of these domains in wild-type led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist sp. strain ATCC 26185 and then expressing them in wild-type and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs.

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“…The general mechanisms of the catalytic domains could be inferred from studies of homologs from FAS and other systems, and the identified active site residues correlate by and large with these expectations 45 . The high-resolution structures are thus most useful for understanding particularities of PKS biosynthesis relative to fatty acid assembly.…”

Section: The Dissection Approach To Studying Functional Domainsmentioning

confidence: 94%

The structural biology of biosynthetic megaenzymes

Weissman¹

2015

Nat Chem Biol

185

165

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The modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are among the largest and most complicated enzymes in nature. In these biosynthetic systems, independently folding protein domains, which are organized into units called 'modules', operate in assembly-line fashion to construct polymeric chains and tailor their functionalities. Products of PKSs and NRPSs include a number of blockbuster medicines, and this has motivated researchers to understand how they operate so that they can be modified by genetic engineering. Beginning in the 1990s, structural biology has provided a number of key insights. The emerging picture is one of remarkable dynamics and conformational programming in which the chemical states of individual catalytic domains are communicated to the others, configuring the modules for the next stage in the biosynthesis. This unexpected level of complexity most likely accounts for the low success rate of empirical genetic engineering experiments and suggests ways forward for productive megaenzyme synthetic biology.

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Structural analysis of protein–protein interactions in type I polyketide synthases

Cited by 37 publications

References 165 publications

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

The structural biology of biosynthetic megaenzymes

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