The effect of divalent cations on the thermostability of type II polyketide synthase acyl carrier proteins

Rivas, Marco A.; Courouble, Valentine V; Baker, Miranda C.; Cookmeyer, David L.; Fiore, Kristen E.; Frost, Alexander J.; Godbe, Kerilyn N.; Jordan, Michael R.; Krasnow, Emily N.; Mollo, Aurelio; Ridings, Stephen T.; Sawada, Keisuke; Shroff, Kavita D.; Studnitzer, Bradley; Thiele, Grace A. R.; Sisto, Ashley; Nawal, Saadia; Huff, Adam R.; Fairman, Robert; Johnson, Karl A.; Beld, Joris; Kokona, Bashkim; Charkoudian, Louise K.

doi:10.1002/aic.16402

Cited by 9 publications

(28 citation statements)

References 50 publications

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“…AcpP N -terminal His 6 -tag, AcpP N -and C -terminal His 6 -tag, and FabF ( C -terminal His 6 -tag) were expressed from pKJ5535 35 or pTL14 36 , and pXY-FabF 36 , respectively. UNIPROT accession numbers for the proteins used in this work are P0A6A8 ( E .…”

Section: Methodsmentioning

confidence: 99%

Colorimetric Assay Reports on Acyl Carrier Protein Interactions

Acheampong

Kokona

Braun

et al. 2019

Sci Rep

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The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4′-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB−) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2−, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial agents by enabling the rapid identification of small molecule inhibitors of ACP-KS interactions.

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Section: Methodsmentioning

confidence: 99%

Colorimetric Assay Reports on Acyl Carrier Protein Interactions

Acheampong

Kokona

Braun

et al. 2019

Sci Rep

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“…After incubation at room temperature (30 min), 10 μL of the ligation product mixture was transformed into chemically competent DH5α cells and plated on LB agar plates (50 μg/mL kanamycin). The plasmids for Arm ACP, Ben ACP, and WhiE ACP were prepared by these means for a previous study 28 . The plasmid for Rat ACP was prepared for this study (see Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Tracking carrier protein motions with Raman spectroscopy

et al. 2019

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Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. Carrier proteins (CPs) play a central role in biosynthesis, but the fast motions of CPs make their conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we present a low-resource method to directly reveal carrier protein-substrate interactions. Chemoenzymatic loading of commercially available, alkyne-containing substrates onto CPs enables rapid visualization of the molecular cargo’s local environment using Raman spectroscopy. This method could clarify the foundations of the chain sequestration mechanism, facilitate the rapid characterization of CPs, and enable visualization of the vectoral processing of natural products both in vitro and in vivo.

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“…Although the sequences of type II PKS ACPs are variable ( 42 ), their overall structure and function are conserved ( Figs. 1 and 3 ) ( 40 ).…”

Section: Sequence Structure and Function Of Type II Pks Acpsmentioning

confidence: 99%

“…3 A , S41); (2) a threonine at the N -terminus of helix IV ( Fig. 3 A , T69); (3) a negative patch of residues connecting helix I and II; (4) a hydrophobic patch in helix IV, and (5) a negative patch of residues in helix III ( 42 ). Type II PKS ACPs are highly acidic, with a net charge at physiological pH estimated to be from −6.3 to −16.2 ( 42 ).…”

Section: Sequence Structure and Function Of Type II Pks Acpsmentioning

confidence: 99%

“…3 A , T69); (3) a negative patch of residues connecting helix I and II; (4) a hydrophobic patch in helix IV, and (5) a negative patch of residues in helix III ( 42 ). Type II PKS ACPs are highly acidic, with a net charge at physiological pH estimated to be from −6.3 to −16.2 ( 42 ). Helices II and III typically harbor 5 to 9 acidic residues and 1 to 3 basic residues, with some acidic residues suspected to be involved in cation binding and/or enzyme partner binding via electrostatic interactions ( 42 ).…”

Section: Sequence Structure and Function Of Type II Pks Acpsmentioning

confidence: 99%

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Probing the structure and function of acyl carrier proteins to unlock the strategic redesign of type II polyketide biosynthetic pathways

Sulpizio¹,

Crawford²,

Koweek³

et al. 2021

Journal of Biological Chemistry

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Type II polyketide synthases (PKSs) are protein assemblies, encoded by biosynthetic gene clusters in microorganisms, that manufacture structurally complex and pharmacologically relevant molecules. Acyl carrier proteins (ACPs) play a central role in biosynthesis by shuttling malonyl-based building blocks and polyketide intermediates to catalytic partners for chemical transformations. Because ACPs serve as central hubs in type II PKSs, they can also represent roadblocks to successfully engineering synthases capable of manufacturing ‘unnatural natural products.’ Therefore, understanding ACP conformational dynamics and protein interactions is essential to enable the strategic redesign of type II PKSs. However, the inherent flexibility and transience of ACP interactions pose challenges to gaining insight into ACP structure and function. In this review, we summarize how the application of chemical probes and molecular dynamic simulations has increased our understanding of the structure and function of type II PKS ACPs. We also share how integrating these advances in type II PKS ACP research with newfound access to key enzyme partners, such as the ketosynthase-chain length factor, sets the stage to unlock new biosynthetic potential.

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The effect of divalent cations on the thermostability of type II polyketide synthase acyl carrier proteins

Cited by 9 publications

References 50 publications

Colorimetric Assay Reports on Acyl Carrier Protein Interactions

Colorimetric Assay Reports on Acyl Carrier Protein Interactions

Tracking carrier protein motions with Raman spectroscopy

Probing the structure and function of acyl carrier proteins to unlock the strategic redesign of type II polyketide biosynthetic pathways

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