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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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Characterization of ACP domains by mass spectrometry.(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.
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pone-0057859-g005: Characterization of ACP domains by mass spectrometry.(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.

Mentions: Functionally competent ACP domains are typically modified by the enzyme-catalyzed transfer of the 4′-phospho-pantetheinyl moiety from coenzyme A (CoA) [11]. In order to assess whether the recombinant ACP fragments are functionally competent, we incubated the purified ACP protein with the sfp phosphopantethinyl transferase (PPTase) from Bacillus subtilis and CoA, and the reaction products were analyzed by LC-MS to determine the molecular weight of the unmodified and modified ACP proteins. Most of the purified tandem-ACP is initially in an unmodified form as evidenced by the measured molecular weight of 59,122 Da, which is close to the molecular weight of 59,116 Da determined by the amino acid sequence (Figure 5A). However, there is a small portion of the protein which shows a change of +322 Da which suggests that some of the protein may have been modified by the endogenous PPTase from the E. coli expression host. The reaction products show a distribution of modified ACP domains in which either one, two, three or four ACP domains were modified as evidenced by their increased molecular weight (Figure 5B). No peak was detected corresponding to the tandem ACP modified on all five domains.


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)

Characterization of ACP domains by mass spectrometry.(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585217&req=5

pone-0057859-g005: Characterization of ACP domains by mass spectrometry.(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.
Mentions: Functionally competent ACP domains are typically modified by the enzyme-catalyzed transfer of the 4′-phospho-pantetheinyl moiety from coenzyme A (CoA) [11]. In order to assess whether the recombinant ACP fragments are functionally competent, we incubated the purified ACP protein with the sfp phosphopantethinyl transferase (PPTase) from Bacillus subtilis and CoA, and the reaction products were analyzed by LC-MS to determine the molecular weight of the unmodified and modified ACP proteins. Most of the purified tandem-ACP is initially in an unmodified form as evidenced by the measured molecular weight of 59,122 Da, which is close to the molecular weight of 59,116 Da determined by the amino acid sequence (Figure 5A). However, there is a small portion of the protein which shows a change of +322 Da which suggests that some of the protein may have been modified by the endogenous PPTase from the E. coli expression host. The reaction products show a distribution of modified ACP domains in which either one, two, three or four ACP domains were modified as evidenced by their increased molecular weight (Figure 5B). No peak was detected corresponding to the tandem ACP modified on all five domains.

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