Fatty Acid Biosynthetic Manipulation in Microalgae Figure 4. Activity-based crosslinking as a determinant of functional interaction with C. reinhardtii cACP. (A) Activity-based substrate mimics used in crosslinking assay; (B) SDS-PAGE gel showing Cr-cACP/CrTE interaction. Apo-Cr-cACP was modified with pantetheine analogue 2 or 3 to generate the corresponding crypto-Cr-cACPs (4 and 5). Crypto-Cr-cACPs were incubated with CrTE and crosslinking was visualized by SDS-PAGE analysis following FLAG affinity purification of the CrACP/TE complex. The band observed at 50 kDa is the FLAG-tagged CrTE and the band detected at ,75 kDa is the ACP/TE complex. During overnight incubation at 37uC, reduced CrTE shows spontaneous oxidation (bands at ,100 kDa). Pantetheine analogues used in crosslinking reactions are noted under the gel. A red arrow illustrates the ACP-TE complex in (B) and (C). (C) Apo-Cr- cACP was reacted with either a-bromopalmitic pantetheine probe 3 or a-bromohexyl pantetheine probe 6 to generate crypto-Cr-cACPs with C16 and C6 acyl chains attached, respectively. Each crypto- Cr-cACP was incubated with CrTE and monitored for extent of crosslinking by SDS-PAGE analysis of Ni-NTA-purified reactions. (D) Apo-Cr-cACP was modified with a-bromopalmitic pantetheine analogue 3 to form crypto-Cr-cACP, which was incubated with UcTE (left 2 lanes) or ChTE (right 2 lanes). Crosslinking was measured by SDS-PAGE analysis following FLAG affinity purification. The bands at ,50 kDa are plant TEs. doi:10.1371/journal.pone.0042949.g004 prokaryotes [43]. The type II FAS iteratively condenses 2-carbon acetate units, each cycle resulting in a fully reduced extended carbon chain. All reactions occur with substrates bound as thioesters to a 49-phosphopantetheine arm attached to ACP. In bacteria, interactions between the ACP and KS are required for proper metabolite assembly [26]. Further studies have established that substrate recognition is also important in KS-mediated condensation [25]. Here, we have shown that significant ACP- TE interactions as well as enzyme-substrate interactions play a role in the hydrolysis of fatty acids within an algal chloroplast. Docking of Cr-cACP to CrTE identifies a protein-protein recognition surface as well as proximity between the fatty-acyl thioester attached to the Cr-cACP and active site cysteine of CrTE (Fig. 2), not dissimilar to ACP/TE docking in Jatropha curcas [29]. To trap transient ACP-TE interactions in vitro, we designed, synthesized, and implemented chemical probes inspired by the enzymatic activity of the TE (Figs. 3C, 3D). Activity-based probe 3 was attached to Cr-cACP to form crypto-Cr-cACP 5 (Fig. 3B) [35], and indeed, a positive functional interaction between CrTE and Cr-cACP triggered a site-specific crosslinking reaction, effectively trapping the algal ACP-TE complex (Fig. 4B). A crosslinked ACP- TE complex was observed between crypto-Cr-cACP and CrTE using both chloroacrylic pantetheine probe 2 and longer chain a- bromopalmitic pantetheine probe 3 (Fig. 3B), demonstrating a positive functional ACP-TE interaction (Fig. 4B). Incubation with longer chain a-bromopalmitic pantetheine probe 3 resulted in full conversion to an ACP-TE complex, whereas chloroacrylic pantetheine probe 2 left unreacted CrTE, demonstrating the specificity of the CrTE probe (Fig. 4B). We did not find any evidence that the racemic nature of our synthesized a-bromo- palmitic pantetheine molecule (3) and its 6 carbon analogue (6) affects the crosslinking reaction, similar to previous studies [26], and there are ongoing efforts in our laboratory to study this further. Although UcTE and ChTE appeared to orient in a similar conformation to Cr-cACP as wildtype CrTE in computational docking studies, no crosslinked complex was detected using all three activity-based probes (2, 3, and 6), indicating that vascular plant TEs did not functionally interact with Cr-cACP in vitro (Figs. 4D, S9). To validate the results of our crosslinking assay in vivo, UcTE and ChTE were engineered into the C. reinhardtii chloroplast (Figs. S11, S12). Although TEs were expressed at relatively high levels (Fig. S12) and enzymatic activity of the TEs was confirmed (Fig. 5), no significant change in fatty acid content or composition was detected in transgenic Cr strains expressing vascular plant TEs, confirming that UcTE and ChTE were unable to hydrolyze fatty acids attached to Cr-cACP due to a lack of molecular recognition. To further demonstrate the specificity of ACP-TE interactions, the Cr mitochondrial ACP (Cr-mACP) did not display favorable binding to CrTE in silico (Fig. S4), as the model of docked Cr- mACP/CrTE predicted a greater distance between the ACP serine and TE cysteine than in the Cr-cACP/CrTE model as well as a different binding orientation that does not appear to facilitate acyl transfer (Fig. S5). This result was validated in vitro, as Cr- mACP did not form a crosslinked complex with CrTE (Fig. S10). The 16 carbon a-bromopantetheinyl-ACP served as a much better substrate mimic than chloroacrylic pantetheinyl-ACP (Fig. 4B). Similar selectivity has been demonstrated with polyketide KS domains that accept elongated hydrocarbon products, indicative of enhanced substrate binding [25]. Since the TE only acts upon a mature fatty-acyl-ACP, we propose that in addition to PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e42949