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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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Domain structure prediction.The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.
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pone-0057859-g003: Domain structure prediction.The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.

Mentions: Initial attempts to identify the boundaries for ACP domains contained in the pfaA multienzyme of Photobacterium profundum, were carried out using the BLASTP tool (NCBI). The BLAST analysis yielded four segments of high similarity to ACP, corresponding to ACP 2,3,4 and 5. To further refine the determination of exact domain boundaries, we used the Udwary-Merski Algorithm (UMA) which assigns a score to each amino acid based on the probability that the amino acid is located within a domain or in an unstructured linker [9]. The UMA analysis of the pfaA sequence reveals areas of high score corresponding to one KS, one AT domain, five ACP domains and one KR domain (Figure 3). One of the five ACP domains detected by UMA, ACP1, had not been identified in the initial BLASTP search, thus highlighting the power of the UMA method in the identification of novel domains [10]. Another ACP domain, ACP5, was found to have a lower UMA score although it was identified in the BLASTP search, possibly suggesting that the fifth ACP domain may not be as conserved or may be more hydrophobic than the other domains. Interestingly, the UMA bioinformatic tool identified a region (residues H1771–R1791) with a high score but a low sequence similarity to ACP, potentially the C-terminal region which enhances biosynthetic activity as described in Gu et al., 2011 [8].


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)

Domain structure prediction.The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057859-g003: Domain structure prediction.The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.
Mentions: Initial attempts to identify the boundaries for ACP domains contained in the pfaA multienzyme of Photobacterium profundum, were carried out using the BLASTP tool (NCBI). The BLAST analysis yielded four segments of high similarity to ACP, corresponding to ACP 2,3,4 and 5. To further refine the determination of exact domain boundaries, we used the Udwary-Merski Algorithm (UMA) which assigns a score to each amino acid based on the probability that the amino acid is located within a domain or in an unstructured linker [9]. The UMA analysis of the pfaA sequence reveals areas of high score corresponding to one KS, one AT domain, five ACP domains and one KR domain (Figure 3). One of the five ACP domains detected by UMA, ACP1, had not been identified in the initial BLASTP search, thus highlighting the power of the UMA method in the identification of novel domains [10]. Another ACP domain, ACP5, was found to have a lower UMA score although it was identified in the BLASTP search, possibly suggesting that the fifth ACP domain may not be as conserved or may be more hydrophobic than the other domains. Interestingly, the UMA bioinformatic tool identified a region (residues H1771–R1791) with a high score but a low sequence similarity to ACP, potentially the C-terminal region which enhances biosynthetic activity as described in Gu et al., 2011 [8].

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