Acyl Carrier Protein Cyanylation Delivers a Ketoacyl Synthase–Carrier Protein Cross-Link

Thiele, Grace A. R.; Friedman, Connie; Tsai, Kathleen J. S.; Beld, Joris; Londergan, Casey H.; Charkoudian, Louise K.

doi:10.1021/acs.biochem.7b00219

Cited by 14 publications

(23 citation statements)

References 17 publications

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“…ACPs were expressed and purified as described previously. 27 In brief, ACP expression plasmids were transformed into chemically competent BAP1 cells for expression. 28 Seed cultures (10 mL LB, 50 μg kanamycin/mL or 100 μg/mL carbenicillin) were grown at 37 °C with shaking and then added to 1 L LB production cultures (50 μg kanamycin/mL or 100 μg/mL carbenicillin).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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The successful engineering of biosynthetic pathways hinges on understanding the factors that influence acyl carrier protein (ACP) stability and function. The stability and structure of ACPs can be influenced by the presence of divalent cations, but how this relates to primary sequence remains poorly understood. As part of a course-based undergraduate research experience, we investigated the thermostability of type II polyketide synthase (PKS) ACPs. We observed an approximate 40 °C range in the thermostability amongst the 14 ACPs studied, as well as an increase in stability (5 – 26 °C) of the ACPs in the presence of divalent cations. Distribution of charges in the helix II-loop-helix III region was found to impact the enthalpy of denaturation. Taken together, our results reveal clues as to how the sequence of type II PKS ACPs relates to their structural stability, information that can be used to study how ACP sequence relates to function.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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“…We later reported that converting the terminal thiol of the Ppant arm to a thiocyanate provides a site-specific vibrational spectroscopic probe for ACPs that reports on the Ppant arm conformational dynamics of the ACP itself 5 . In the presence of a compatible KS, the thiocyanate-labeled ACP is activated to form a mechanistically relevant cross-link through disulfide bond formation between the Ppant arm thiol and partner KS thiolate 6 . The concomitant release of CN − enables the monitoring of ACP-KS interactions via infrared (IR) spectroscopy (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Each student designed, constructed, expressed and purified a distinct mutant ACP to convert to the thiocyanate version and assess via mechanistic cross-linking. In addition, students expressed and purified wild type AcpP as a positive control along with the ACP from the actinorhodin type II PKS (ACT ACP) as a negative control since this protein does not bind to FabF in vitro 6–8 . One student observed that their reaction turned yellow upon mixing their thiocyanate-labeled AcpP mutant with the KS.…”

Section: Introductionmentioning

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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“…Due to the dynamic nature of both the protein and PPant 10 , AcpP can sequester diverse cargo lengths and moieties with important cargoinduced structural and regulatory consequences 11 . Synthetic cargo attachment offers an excellent handle for structural biology studies 12,13 . During interaction with partner proteins, the cargo completely translocates from the hydrophobic core of AcpP into the partner in a process called "chain flipping" 14,15 .…”

mentioning

confidence: 99%

Molecular basis for interactions between an acyl carrier protein and a ketosynthase

Milligan

Jackson

et al. 2019

Nat Chem Biol

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Fatty acid synthases are dynamic ensembles of enzymes that can efficiently biosynthesize long hydrocarbon chains. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and β-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and MD simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli. The E. coli fatty acid synthase (FAS) produces fatty acids through an iterative cycle via the

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Acyl Carrier Protein Cyanylation Delivers a Ketoacyl Synthase–Carrier Protein Cross-Link

Cited by 14 publications

References 17 publications

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

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

Colorimetric Assay Reports on Acyl Carrier Protein Interactions

Molecular basis for interactions between an acyl carrier protein and a ketosynthase

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