Structural and Functional Analysis of the Loading Acyltransferase from Avermectin Modular Polyketide Synthase

Wang, Fen; Wang, Yanjie; Ji, Junjie; Zhou, Zhan; Yu, Jing; Zhu, HJ; Su, Zhiguo; Zhang, Lixin; Zheng, Jianting

doi:10.1021/cb500873k

Cited by 43 publications

(52 citation statements)

References 41 publications

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“…4a,b). By contrast, the excised loading AT domain of the avermectin modPKS (AVES1) 28 , which features completely different connectivity via an ACP to the downstream KS domain, has a distinct linker architecture resembling the post-MAT linker in PKS and FAS condensing regions (Supplementary Fig. 4c,d).…”

Section: Resultsmentioning

confidence: 99%

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

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Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

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Section: Resultsmentioning

confidence: 99%

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

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“…B )Structure of the avermectin AT loading domain Ave-AT° (PDB 4RL1). 19 The two teal helices indicate a change in orientation compared to αJ and αP in the SAT structure and the active site S120 is shown as a stick. C ) Electrostatic surface representation of the SAT domain showing a cross section of the cavity leading to the active site and bound hexanoyl.…”

Section: Figurementioning

confidence: 99%

Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis

et al. 2015

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Modular collaboration between iterative fungal polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here, we reconstituted the modular biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM's SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that is also produced by CazF and required in later stages of biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site. The crystal structure of SAT domain will enable protein engineering efforts aimed at mixing and matching different IPKS modules for the biosynthesis of new compounds.

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“…MAT was further truncated by removing LD, based on the X-ray crystal structure of the avermectin loading AT. 31 15 The KS-domain in constructs with (11) or without LD (10) was hardly expressed in E. coli (see Fig. 4C and D).…”

Section: Engineering Of the Mfas Condensing Partmentioning

confidence: 99%

“…DEBS and AVES provide a simple solution for such a design, and feature single loading didomains (AT-ACP didomain) connected to the first module ("module 1"). 31,40 Another solution may be derived from the Pikromycin synthase (PikA), which uses a modified α-module (KS, AT and ACP) with a decarboxylating KS domain and an AT domain, specific for extender substrates. 41,42 Both architectures were considered in our approach of engineering bimodular constructs with either mFAS sequences alone or including loading domains from modular PKSs.…”

Section: Generation Of Bimodular Constructsmentioning

confidence: 99%

Probing the Modularity of Megasynthases by Rational Engineering of a Fatty Acid Synthase (FAS) Type I

Rittner

Paithankar

Drexler

et al. 2018

Preprint

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Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities ("modules") facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity.Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, is of utmost importance for harnessing megasynthases for the synthesis of designer compounds. In this study, we exploit the close semblance between PKSs and animal FAS to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by structural and sequence information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel engineered modules resemble all four common module types of PKSs and demonstrate that this approach can be a powerful tool to create higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design. 3 Keywords Multidomain protein, modularity of megasynthases, polyketide synthase, protein engineering Abbreviations FAS, fatty acid synthase; PKS, polyketide synthases; CMN, Corynebacteria,

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Structural and Functional Analysis of the Loading Acyltransferase from Avermectin Modular Polyketide Synthase

Cited by 43 publications

References 41 publications

The structural organization of substrate loading in iterative polyketide synthases

The structural organization of substrate loading in iterative polyketide synthases

Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis

Probing the Modularity of Megasynthases by Rational Engineering of a Fatty Acid Synthase (FAS) Type I

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