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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.

Show MeSH
Evolutionary conservation of the tandem ACP domain.While the tandem ACP arrangement is rare among most PKS enzyme systems, it is a defining feature of the marine PUFA synthase multienzymes. Most PUFA producers contain between 2–9 ACP domains in tandem.
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pone-0057859-g002: Evolutionary conservation of the tandem ACP domain.While the tandem ACP arrangement is rare among most PKS enzyme systems, it is a defining feature of the marine PUFA synthase multienzymes. Most PUFA producers contain between 2–9 ACP domains in tandem.

Mentions: The ACP domains in the PUFA synthase clusters are arranged in tandem within the pfaA multidomain protein. This rare organization of enzyme domains in the PUFA synthase multienzyme is globally conserved among organisms that produce PUFAs in deep-sea environments [4], [5]. Numerous species of Shewanella, Psychromonas and Nostoc have been found to produce fatty acids in very high yields and to contain PUFA synthase genes organized as outlined in Figure 1. Although the general organization of domains in the PUFA gene cluster is conserved in different organisms, there is one area of variability: the number of ACP domains repeated in tandem along the sequence of pfaA, which tends to oscillate between 2 and 9 copies (Figure 2).


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)

Evolutionary conservation of the tandem ACP domain.While the tandem ACP arrangement is rare among most PKS enzyme systems, it is a defining feature of the marine PUFA synthase multienzymes. Most PUFA producers contain between 2–9 ACP domains in tandem.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057859-g002: Evolutionary conservation of the tandem ACP domain.While the tandem ACP arrangement is rare among most PKS enzyme systems, it is a defining feature of the marine PUFA synthase multienzymes. Most PUFA producers contain between 2–9 ACP domains in tandem.
Mentions: The ACP domains in the PUFA synthase clusters are arranged in tandem within the pfaA multidomain protein. This rare organization of enzyme domains in the PUFA synthase multienzyme is globally conserved among organisms that produce PUFAs in deep-sea environments [4], [5]. Numerous species of Shewanella, Psychromonas and Nostoc have been found to produce fatty acids in very high yields and to contain PUFA synthase genes organized as outlined in Figure 1. Although the general organization of domains in the PUFA gene cluster is conserved in different organisms, there is one area of variability: the number of ACP domains repeated in tandem along the sequence of pfaA, which tends to oscillate between 2 and 9 copies (Figure 2).

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