Reversible labeling of native and fusion-protein motifs

The acyl carrier protein (ACP) requires post-translational modification with a 4’-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-acyl carrier protein synthetases (AasSs) from different organisms are able to load even, odd and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.

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“…AcpH was used to convert apo/holo-carrier protein mixtures into pure apo samples. (Kosa, et al, 2012)…”

Section: Methodsmentioning

confidence: 99%

Versatility of Acyl-Acyl Carrier Protein Synthetases

Beld

Finzel

Burkart

2014

Chemistry & Biology

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“…[48] Both resin-attached and free protein techniques have been developed, allowing for the preparation of large quantities of apo -ACP and the recycling of high-value, isotopically enriched ACPs for NMR use. [49,50] Reversible tagging has also allowed for quantitative “apo-fication” of holo - and crypto-ACPs, yielding homogenous apo -ACP for further modification (Figure 2b). …”

Section: The Acyl Carrier Protein – An Extended Toolkitmentioning

confidence: 99%

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

2015

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Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory.

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“…3 In addition, we recently introduced the use of an ACP hydrolase (AcpH) to cleave PPant and PPant analogues from ACP as a reversible labeling tool, thus providing a full suite of ACP modifying methodologies. 4 Here we further coordinate this dual enzyme utility as immobilized biocatalytic tools, thereby streamlining their application for protein labeling, unlabeling, and isolation.…”

Section: Introductionmentioning

confidence: 99%

“…12 We further fused this recombinant protein with various MBP and His-Tagged fusions and demonstrated activity upon a variety of modified PPant analogues, including fluorescent and natural product mimics. 4 We focus on the C-terminal His-tagged construct as the most versatile form.…”

Section: Introductionmentioning

confidence: 99%

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Resin supported acyl carrier protein labeling strategies

2014

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The post-translational modifying enzymes phophopantetheinyl transferase and acyl carrier protein hydrolase have shown utility in the functional modification of acyl carrier proteins. Here we develop these tools as immobilized biocatalysts on agarose supports. New utility is imparted through these methods, enabling rapid and label-independent protein purification. Immobilization of acyl carrier protein is also demonstrated for rapid activity assays of these 4'-phosophopantetheine modifying enzymes, displaying a particular advantage in the case of phosphopantetheine removal, where few orthogonal techniques have been demonstrated. These tools further enrich the suite of functional utility of 4'-phosophopantetheine chemistry, with applications to protein functionalization, materials, and natural product biosynthetic studies.

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Reversible labeling of native and fusion-protein motifs

Cited by 42 publications

References 30 publications

Versatility of Acyl-Acyl Carrier Protein Synthetases

Versatility of Acyl-Acyl Carrier Protein Synthetases

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

Resin supported acyl carrier protein labeling strategies

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