Structure–Function Analysis of the Extended Conformation of a Polyketide Synthase Module

Li, Xiuyuan; Sevillano, Natalia; Greca, Florencia La; Deis, L.N.; Liu, Yu Chen; Deller, Marc C.; Mathews, I.I.; Matsui, Tsutomu; Cane, David E.; Craik, Charles S.; Khosla, Chaitan

doi:10.1021/jacs.8b02100

Cited by 40 publications

(63 citation statements)

References 20 publications

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“…As compared to the monomeric character of the excised SAT of CazM this represents another example for the relevance of avidity in multienzymes for oligomerization (see above). The condensing region (KS-MAT) reveals the same conserved organization as observed in other PKSs and mFASs 4, 30, 39, 67, 75, 77, 131 , except for PikAIII 76 . The cavity formed by the S-shaped condensing region (view along the dimeric axis) is closed at the bottom by the SATs.…”

Section: Non-reducing Iterative Polyketide Synthasesmentioning

confidence: 60%

The architectures of iterative type I PKS and FAS

2018

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Covering: up to mid of 2018 Type I fatty acid synthases (FASs) are giant multienzymes catalyzing all steps of the biosynthesis of fatty acids from acetyl- and malonyl-CoA by iterative precursor extension. Two strikingly different architectures of FAS evolved in yeast (as well as in other fungi and some bacteria) and metazoans. Yeast-type FAS (yFAS) assembles into a barrel-shaped structure of more than 2 MDa molecular weight. Catalytic domains of yFAS are embedded in an extensive scaffolding matrix and arranged around two enclosed reaction chambers. Metazoan FAS (mFAS) is a 540 kDa X-shaped dimer, with lateral reaction clefts, minimal scaffolding and pronounced conformational variability. All naturally occurring yFAS are strictly specialized for the production of saturated fatty acids. The yFAS architecture is not used for the biosynthesis of any other secondary metabolite. On the contrary, mFAS is related at the domain organization level to major classes of polyketide synthases (PKSs). PKSs produce a variety of complex and potent secondary metabolites; they either act iteratively (iPKS), or are linked via directed substrate transfer into modular assembly lines (modPKSs). Here, we review the architectures of yFAS, mFAS, and iPKSs. We rationalize the evolution of the yFAS assembly, and provide examples for re-engineering of yFAS. Recent studies have provided novel insights into the organization of iPKS. A hybrid crystallographic model of a mycocerosic acid synthase-like Pks5 yielded a comprehensive visualization of the organization and dynamics of fully-reducing iPKS. Deconstruction experiments, structural and functional studies of specialized enzymatic domains, such as the product template (PT) and the starter-unit acyltransferase (SAT) domain have revealed functional principles of non-reducing iterative PKS (NR-PKSs). Most recently, a six-domain loading region of an NR-PKS has been visualized at high-resolution together with cryo-EM studies of a trapped loading intermediate. Altogether, these data reveal the related, yet divergent architectures of mFAS, iPKS and also modPKSs. The new insights highlight extensive dynamics, and conformational coupling as key features of mFAS and iPKS and are an important step towards collection of a comprehensive series of snapshots of PKS action.

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Section: Non-reducing Iterative Polyketide Synthasesmentioning

confidence: 60%

The architectures of iterative type I PKS and FAS

2018

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“…Thus, such an inert F ab could be potentially developed into a tool for dynamic spectroscopic analysis or as a structural fiducial marker in electron microscopy. 31 Together with 1B2, 11 we have put forth two valuable and orthogonal F ab s for structural and enzymological studies of DEBS. This discovery strategy is readily applicable to other complex multidomain enzyme systems.…”

Section: Discussionmentioning

confidence: 99%

“…The F ab –TE complex was produced in a manner similar to a previously reported procedure. 11 3A6 and TE were mixed in a 1:1 ratio for 60 min at 4 °C, followed by purification using a Superdex 200 10/300 size exclusion chromatography (SEC) column (GE Healthcare) pre-equilibrated with SEC buffer. Fractions corresponding to the complex were pooled and concentrated to 4.6 mg/mL.…”

Section: Methodsmentioning

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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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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“…The choice of M3 and M6 was guided by their differing turnover rates with M1 22 and the previously observed substrate restrictions. 16 X-ray structural data exists for KS3-AT3, 36,37 which gave us the opportunity to run the FuncLib method for the calculation of stable active sites on authentic data. For KS6-AT6, a homology model was generated on the basis of a sequence similarity of ~55% to the structurally solved KS-AT didomains KS3-AT3 36 and KS5-AT5 19 and the model was also submitted to FuncLib.…”

Section: Results and Discussion Design Of Ks Multipoint Mutants-mentioning

confidence: 99%

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

Klaus

Buyachuihan

Grininger

2020

Preprint

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Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the non-cognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the ACP. Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail.

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Structure–Function Analysis of the Extended Conformation of a Polyketide Synthase Module

Cited by 40 publications

References 20 publications

The architectures of iterative type I PKS and FAS

The architectures of iterative type I PKS and FAS

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

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

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