Multienzyme docking in hybrid megasynthetases

Richter, Carsten D.; Nietlispach, Daniel; Broadhurst, R. William; Weissman, Kira J.

doi:10.1038/nchembio.2007.61

Cited by 87 publications

(139 citation statements)

References 47 publications

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“…Hybrid PKS-NRPSs are remarkable macromolecular assembly lines with carrier proteins delivering substrates from one enzyme module to the next and docking domains providing the intermodular communication that allows for the proper directionality (14,25,26). Our structures provide a visualization of the interactions between an N-terminal docking domain and a downstream enzyme within a NRPS module, and we find a bead-on-a-string type arrangement.…”

Section: Discussionmentioning

confidence: 94%

“…S3D). This difference in the β-hairpin is interesting given that this region is predicted to be a binding site of the cognate recognition sequence (14,25). However, it is not currently clear if theses variations are mechanistically significant or an artifact of a difference in oligomeric state.…”

Section: Resultsmentioning

confidence: 99%

“…This docking domain functions at an NRPS-NRPS junction and shares with EpoBdd 33% sequence identity and an RMSD of 3.6 Å for 52 Cα atoms (Fig. S3) (14). The β-hairpin and the third α-helix (α3′) of the αββαα fold show the largest deviation between these two docking domain structures (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2A), and is connected to the Cy domain by a 20-residue linker (L56-T75). There is one other docking domain structure with this fold, TubCdd from the Angiococcus disciformitus tubulysin system (14). This docking domain functions at an NRPS-NRPS junction and shares with EpoBdd 33% sequence identity and an RMSD of 3.6 Å for 52 Cα atoms (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequent oxidation by the flavin-dependent oxidase domain (EpoB-Ox) results in 2-methylthiazole (8,10,11). PKS-NRPS docking domains (14), referred to as EpoAdd and EpoBdd for the upstream donor and the downstream acceptor proteins, respectively, serve to localize the T domains to the appropriate intermodule junction, facilitating what has often been referred to as an assembly line biosynthetic process.…”

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confidence: 99%

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Structural elements of an NRPS cyclization domain and its intermodule docking domain

Dowling

Kung

Croft

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.crystal structure | molecular dynamics | epothilone | natural product

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Dowling

Kung

Croft

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Biosynthesis of Pharmaceutical Natural Products and Their Pathway Engineering

Smanski

Liu

et al. 2012

Organic Chemistry – Breakthroughs and Perspectives

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DNA as a Recyclable Natural Polymer

Liu

Giaveri

Ortiz

et al. 2022

Adv Funct Materials

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Nature has the ability of circularly re-using its components to produce the molecules and materials it needs. An example is the ability of most living organisms of digesting proteins they feed off into amino acids and then using such amino acids in the ribosomal synthesis of new proteins. Recently, it has been shown that such recycling of proteins can be reproduced outside living organisms. The key proteins' feature that allows for this type of recycling is their being sequence-defined polymers. Arguably, nature's most famous sequencedefined polymer is DNA. Here it is shown that it is possible, starting from sheared calf-DNA, to obtain all four nucleotides as monophosphate-nucleotides (dNMPs). These dNMPs are phosphorylated in a one-pot, multi-enzymes, phosphorylation reaction to generate triphosphate-nucleotides (dNTPs). Finally, dNTPs so achieved (with a global yield of ∼60%) are used as reagents for PCR (polymerase chain reaction) to produce target DNA strands, and for the diagnosis of targeted DNA by quantitative PCR (qPCR). This approach is an efficient, convenient, and environmentally friendly way to produce dNTPs and DNA through recycling according to the paradigm of a circular economy.

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Multienzyme docking in hybrid megasynthetases

Cited by 87 publications

References 47 publications

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Biosynthesis of Pharmaceutical Natural Products and Their Pathway Engineering

DNA as a Recyclable Natural Polymer

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