Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

Barajas, Jesus F.; Finzel, Kara; Valentic, Timothy R.; Shakya, Gaurav; Gamarra, Nathan; Martinez, Delsy; Meier, Jordan L.; Vagstad, Anna L.; Newman, Adam G.; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou‐Chuan

doi:10.1002/anie.201605401

Cited by 16 publications

(11 citation statements)

References 21 publications

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“…In accordance with literature precedent (39,47,49,(62)(63)(64)(65), we investigated PPIs for AcpP-FabD by comparing the crosslinking efficiencies of a panel of FabD S92C interface mutants with AcpP loaded with a C2-α-bromo-pantetheinamide probe (SI Appendix, Figs. S10 B and C and S11).…”

Section: Resultsmentioning

confidence: 99%

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Misson

Mindrebo

Davis

et al. 2020

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

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Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP–AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP–AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP–AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP–AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

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Section: Resultsmentioning

confidence: 99%

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Misson

Mindrebo

Davis

et al. 2020

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

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“…Probe 54 has also been used to characterize interactions between ACPs with the structurally and functionally related product template (PT) domains discussed further in section 3.4 . 112…”

Section: Chemical Tools To Interrogate Pks Enzymesmentioning

confidence: 99%

Trapping interactions between catalytic domains and carrier proteins of modular biosynthetic enzymes with chemical probes

Gulick

Aldrich

2018

Nat. Prod. Rep.

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Covering: up to early 2018 The Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) are families of modular enzymes that produce a tremendous diversity of natural products, with antibacterial, antifungal, immunosuppressive, and anticancer activities. Both enzymes utilize a fascinating modular architecture in which the synthetic intermediates are covalently attached to a peptidyl- or acyl-carrier protein that is delivered to catalytic domains for natural product elongation, modification, and termination. An investigation of the structural mechanism therefore requires trapping the often transient interactions between the carrier and catalytic domains. Many novel chemical probes have been produced to enable the structural and functional investigation of multidomain NRPS and PKS structures. This review will describe the design and implementation of the chemical tools that have proven to be useful in biochemical and biophysical studies of these natural product biosynthetic enzymes.

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“…Among the six proteins encoded, enzymes Bik1, Bik2, and Bik3 are predicted to be responsible for the synthesis of bikaverin, while Bik4 and Bik5 are predicted transcription regulators, and Bik6 is a permease 18 . The polyketide carbon-skeleton of bikaverin is produced by the Type I PKS Bik1 (formerly called PKS4), which is a very large (over 2000 amino acid residues) multifunctional enzyme consisting of the following domains: starter unit acyltransferase (SAT), β-ketosynthase (KS), malonyl:ACP acyltransferase (MAT), product template (PT), acyl carrier protein (ACP), and thioesterase/claisen cyclase (TE/CLC) 20 , 21 . The SAT domain initially selects acetyl-CoA as a starting unit, and the KS and MAT domains condense eight units of malonyl-CoA to the growing polyketide chain, resulting in a polyketide chain, which is covalently tethered to the ACP domain throughout chain elongation.…”

Section: Introductionmentioning

confidence: 99%

Pathway engineering in yeast for synthesizing the complex polyketide bikaverin

Zhao

Yao

et al. 2020

Nat Commun

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Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.

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Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

Cited by 16 publications

References 21 publications

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Trapping interactions between catalytic domains and carrier proteins of modular biosynthetic enzymes with chemical probes

Pathway engineering in yeast for synthesizing the complex polyketide bikaverin

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