Mapping the catalytic conformations of an assembly-line polyketide synthase module

Cogan, D.P.; Zhang, Kaiming; Li, Xiuyuan; Li, Shanshan; Pintilie, Grigore; Roh, Sook Young; Craik, Charles S.; Chiu, Wah; Khosla, Chaitan

doi:10.1126/science.abi8358

Cited by 58 publications

(97 citation statements)

References 69 publications

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“…This work revealed that interdomain contacts are critical for establishing the various functional states of the module, and that transitions between such states rely on evolving interfaces between the domains, as well as the intervening ‘linker’ regions. This view of modular function was recently reinforced by cryo-EM/crystallographic analysis of additional modules sourced from two PKS systems 6 , 7 . In short, PKS modules appear to be highly integrated units, thus explaining why exchange of catalytic domains for heterologous counterparts is often detrimental 8 .…”

Section: Introductionmentioning

confidence: 95%

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

et al. 2022

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The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites − a reduction in chain length of 14 carbons relative to the 51-membered parental compounds − but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

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Section: Introductionmentioning

confidence: 95%

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

et al. 2022

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“…Because 1B2 did not inhibit the core catalytic activities of the module, it supported the hypothesis that the “extended” conformation is preserved throughout the catalytic cycle. Two recent Cryo-EM studies further underscore this hypothesis, both studies leveraged 1B2 and found the F ab to be crucial in their analysis [ 61 , 62 ]. The Lasalocid A PKS module (Lsd14) was determined at 2.4 (X-ray) and 3.1 Å (Cryo-EM) resolutions, respectively.…”

Section: Polyketide Synthases (Pkss)mentioning

confidence: 99%

“…Identification of neutral F ab s that bind different PKS epitopes could help resolve portions of Cryo-EM models that remain elusive. One portion of the PKS that remains especially difficult to model is the flexible TE domain [ 61 ]. The structure of a F ab that recognizes the DEBS TE (3A6, PDB ID 6MLK ) has been reported; this can be considered a neutral F ab because the TE maintains its catalytic activity as part of the complex [ 61 , 63 ].…”

Section: Polyketide Synthases (Pkss)mentioning

confidence: 99%

“…One portion of the PKS that remains especially difficult to model is the flexible TE domain [ 61 ]. The structure of a F ab that recognizes the DEBS TE (3A6, PDB ID 6MLK ) has been reported; this can be considered a neutral F ab because the TE maintains its catalytic activity as part of the complex [ 61 , 63 ]. As previously reported ( Fig.…”

Section: Polyketide Synthases (Pkss)mentioning

confidence: 99%

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Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases

Guzman

Khosla

2022

Synthetic and Systems Biotechnology

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The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (F ab s) as protein crystallization chaperones is discussed. F ab s have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three F ab s onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying F ab s to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One F ab in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3–5 Å) resolution. The identification of PKS-binding F ab s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

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“…These complex structures provide detailed molecular insights into how trans-ATs recognize their ACP partners. During the preparation of this manuscript, structures of cis-AT PKS modules were reported (Bagde et al, 2021;Cogan et al, 2021). The X-ray structure of Lsd14 shows the transacylation state of apo LsdACP7.…”

Section: Introductionmentioning

confidence: 99%

Structural visualization of transient interactions between the cis-acting acyltransferase and acyl carrier protein of the salinomycin modular polyketide synthase

Feng¹,

Zhang²,

Huang³

et al. 2022

Acta Crystallogr D Struct Biol

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Transient protein–protein interactions between cis-acting acyltransferase (AT) and acyl carrier protein (ACP) domains are critical for the catalysis and processivity of modular polyketide synthases (mPKSs), but are challenging for structural characterization due to the intrinsically weak binding affinity. Here, a stable complex of cis-acting AT and ACP domains from the ninth module of the salinomycin mPKS was obtained using a maleimide cross-linker and the structure of the complex was determined at 2.6 Å resolution. The crystal structure shows that the AT in combination with the ketosynthase (KS)-to-AT linker forms a C-shaped architecture to embrace the ACP. The large hydrolase subdomain of the AT serves as a major binding platform for the ACP, while the small ferredoxin-like subdomain of the AT and the KS-to-AT linker cooperate with each other to constrain binding of the ACP. The importance of interface residues in cis-acting AT–ACP interactions was confirmed by mutagenesis assays. The interaction mode observed in the cis-acting AT–ACP complex is completely different from those observed in trans-acting AT–ACP complexes, where the ACP primarily contacts the small domain of the AT. The complex structure provides detailed mechanistic insights into AT–ACP recognition in cis-AT mPKSs.

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Mapping the catalytic conformations of an assembly-line polyketide synthase module

Cited by 58 publications

References 69 publications

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases

Structural visualization of transient interactions between the cis-acting acyltransferase and acyl carrier protein of the salinomycin modular polyketide synthase

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