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Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

Trujillo U, Vázquez-Rosa E, Oyola-Robles D, Stagg LJ, Vassallo DA, Vega IE, Arold ST, Baerga-Ortiz A - PLoS ONE (2013)

Bottom Line: However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit.Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein.Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

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The polyunsaturated fatty acid synthase domain structure.A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).
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pone-0057859-g001: The polyunsaturated fatty acid synthase domain structure.A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).

Mentions: Polyunsaturated fatty acids (PUFAs) are made by deep-sea bacteria employing an anaerobic mechanism that involves a polyketide synthase (PKS)-like multienzyme [1], [2]. In this multienzyme system, a total of five genes have been found to be required for the production of PUFA (Figure 1): pfaA which contains sequences corresponding to a ketoacyl synthase (KS) domain, an acyltransferase (AT) domain, a stretch containing multiple acyl carrier protein (ACP) domains and finally a ketoreductase (KR) domain; pfaB consists of a single AT domain; pfaC contains two KS homology domains and two DH domains; pfaD contains a single enoyl reductase (ER) domain and pfaE encodes for a phosphopantetheinyl transferase which is typically required for the activation of the ACP domains [3], [4]. While some work has been carried out on individual activities within this multienzyme system, the actual mechanism by which the enzymes act in concert to produce PUFAs has not been elucidated.


Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

Trujillo U, Vázquez-Rosa E, Oyola-Robles D, Stagg LJ, Vassallo DA, Vega IE, Arold ST, Baerga-Ortiz A - PLoS ONE (2013)

The polyunsaturated fatty acid synthase domain structure.A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585217&req=5

pone-0057859-g001: The polyunsaturated fatty acid synthase domain structure.A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).
Mentions: Polyunsaturated fatty acids (PUFAs) are made by deep-sea bacteria employing an anaerobic mechanism that involves a polyketide synthase (PKS)-like multienzyme [1], [2]. In this multienzyme system, a total of five genes have been found to be required for the production of PUFA (Figure 1): pfaA which contains sequences corresponding to a ketoacyl synthase (KS) domain, an acyltransferase (AT) domain, a stretch containing multiple acyl carrier protein (ACP) domains and finally a ketoreductase (KR) domain; pfaB consists of a single AT domain; pfaC contains two KS homology domains and two DH domains; pfaD contains a single enoyl reductase (ER) domain and pfaE encodes for a phosphopantetheinyl transferase which is typically required for the activation of the ACP domains [3], [4]. While some work has been carried out on individual activities within this multienzyme system, the actual mechanism by which the enzymes act in concert to produce PUFAs has not been elucidated.

Bottom Line: However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit.Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein.Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Show MeSH