Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis

Argyropoulos, Panos; Bergeret, Fabien; Pardin, Christophe; Reimer, Janice M; Pinto, Atahualpa; Boddy, Christopher N.; Schmeing, T.M.

doi:10.1016/j.bbagen.2015.11.007

Cited by 17 publications

(21 citation statements)

References 43 publications

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“…Antibody 3A6 was bound principally through extensive hydrogen bonding to a structured region on the TE surface (Figure 5B) that is distant from the previously reported tunnel through which the substrate and product are thought to enter and exit, respectively, the active site. 21,29,30 Variable regions from both the heavy chain and light chain fragments contributed to interaction with TE (Figure 5B,C). Its binding to the TE appeared to represent an example of lock-and-key recognition, as no significant rearrangements were observed in the TE backbone as a result of 3A6 binding (Figure 6A; root-mean-square deviation of 0.359 Å).…”

Section: Resultsmentioning

confidence: 98%

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Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Li¹,

Sevillano

Greca

et al. 2018

Biochemistry

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Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a Fab (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE–3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein–protein recognition mechanism. Fab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.

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Section: Resultsmentioning

confidence: 98%

“…3A6 binding induces minimal structural distortion in the TE structure. (A) Superimposed structures of the TE from the antibody–TE complex (blue) and the TE alone (cyan, PDB entry 5D3K 30 ). The root-mean-square deviation = 0.359 Å.…”

Section: Figurementioning

confidence: 99%

Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Li¹,

Sevillano

Greca

et al. 2018

Biochemistry

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“…Several TE domains from NRPS and PKS systems have been structurally characterized 58,[88][89][90][91][92]153,182,183 (Figure 9b). They show the α/β hydrolase fold, the active site triad and a putative oxyanion hole to stabilize the negatively charged tetrahedral transition state.…”

Section: F I G U R E 8 Proposed Asperphenamate Biosynthesis Based Onmentioning

confidence: 99%

“…Attempts to generate intermediate‐bound acyl‐ O ‐TE structures, representative of the product of the first half reaction and the substrate of the second half reaction, are hampered by the relative short lifetime of the acyl ester intermediates 185,186 . A few structures of TE‐substrate complexes exist, using phosphonate analogues 182,183,187 or employing an expanded genetic code strategy to generate stable amide intermediates 153 (discussed in Section 5.2.2.1). In the analog‐bound structures of the pikromycin TE bound to a pentaketide phosphonate, 187 the linear pikromycin intermediate curls back in the cavity between the lid and active site toward the catalytic Ser.…”

Section: Depsipeptides With Ester Bonds Formed During Terminationmentioning

confidence: 99%

Biosynthesis of depsipeptides, or Depsi: The peptides with varied generations

Alonzo

Schmeing

2020

Protein Science

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Depsipeptides are compounds that contain both ester bonds and amide bonds. Important natural product depsipeptides include the piscicide antimycin, the K + ionophores cereulide and valinomycin, the anticancer agent cryptophycin, and the antimicrobial kutzneride. Furthermore, database searches return hundreds of uncharacterized systems likely to produce novel depsipeptides. These compounds are made by specialized nonribosomal peptide synthetases (NRPSs). NRPSs are biosynthetic megaenzymes that use a module architecture and multi-step catalytic cycle to assemble monomer substrates into peptides, or in the case of specialized depsipeptide synthetases, depsipeptides. Two NRPS domains, the condensation domain and the thioesterase domain, catalyze ester bond formation, and ester bonds are introduced into depsipeptides in several different ways. The two most common occur during cyclization, in a reaction between a hydroxy-containing side chain and the C-terminal amino acid residue in a peptide intermediate, and during incorporation into the growing peptide chain of an α-hydroxy acyl moiety, recruited either by direct selection of an α-hydroxy acid substrate or by selection of an α-keto acid substrate that is reduced in situ. In this article, we discuss how and when these esters are introduced during depsipeptide synthesis, survey notable depsipeptide synthetases, and review insight into bacterial depsipeptide synthetases recently gained from structural studies.

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“…Finally, it is not yet clear whether some combination of sequence and structure could be used to predict macrocyling TEs from TEs that simply catalyze hydrolysis. Efforts to co-crystallize a TE with macrocycle-like inhibitors are ongoing and will undoubtedly reveal valuable insights once successful [ 94 ]. TEs have also been assayed with various substrates as an empirical way of elucidating their recognition features.…”

Section: Thioesterases: Determinants Of Macrocylizationmentioning

confidence: 99%

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

Bayly

Yadav

2017

Molecules

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Modular polyketide synthases (mPKSs) build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters (modules) of independently-folding domains, these assembly-line-like megasynthases can be engineered by introducing non-native components. However, poor introduction points and incompatible domain combinations can cause both unintended products and dramatically reduced activity. This limits the engineering and combinatorial potential of mPKSs, precluding access to further potential therapeutics. Different regions on a given mPKS domain determine how it interacts both with its substrate and with other domains. Within the assembly line, these interactions are crucial to the proper ordering of reactions and efficient polyketide construction. Achieving control over these domain functions, through precision engineering at key regions, would greatly expand our catalogue of accessible polyketide products. Canonical mPKS domains, given that they are among the most well-characterized, are excellent candidates for such fine-tuning. The current minireview summarizes recent advances in the mechanistic understanding and subsequent precision engineering of canonical mPKS domains, focusing largely on developments in the past year.

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Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis

Cited by 17 publications

References 43 publications

Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Biosynthesis of depsipeptides, or Depsi: The peptides with varied generations

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

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