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

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

Multiple sequence alignment of the five ACP domains.

<p>The ACP domains from the <i>Photobacterium profundum</i> PUFA synthase were aligned using ClustalW. The black bars denote the stretches of protein sequence predicted to be α-helices.</p

Expected and Measured Molecular Weights of ACP Constructs in Solution.

<p>Expected and Measured Molecular Weights of ACP Constructs in Solution.</p

Characterization of ACP domains by mass spectrometry.

<p>(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.</p

Oligonucleotides used for the amplification of the different proteins in this study.

<p>Oligonucleotides used for the amplification of the different proteins in this study.</p

Domain structure prediction.

<p>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.</p

Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b>

<p>Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b></p

Solution structure of tandem ACP.

<p>(A) The three-dimensional bead model constructed by ‘dammif’ reveals a molecular volume of 96,600 ų. (B) Simulation of the scattering data based on structural models reveals that an extended and flexible overall configuration can sufficiently account for the observed data. Figures A and B are on a different scale.</p

The polyunsaturated fatty acid synthase domain structure.

<p>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).</p

Input parameters for the UMA calculations.

<p>Input parameters for the UMA calculations.</p