LETTER
Mechanism-Based Protein Cross-Linking Probes To Investigate Carrier ProteinMediated Biosynthesis
Andrew S. Worthington, Heriberto Rivera, Jr., Justin W. Torpey, Matthew D. Alexander, and Michael D. Burkart* Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358
T
he carrier protein is a highly conserved, small (⬃10 kDa), acidic domain found in fatty acid, polyketide, and nonribosomal peptide synthases. Whether incorporated into a large multidomain megasynthase (type I systems) or as isolated proteins functioning independently (type II systems), carrier proteins are highly flexible proteins (1, 2). Because of inherent technical hurdles posed by the cloning and expression of large, multidomain synthases, carrier protein participation within these systems remains largely uncharacterized, and those systems that have been studied show disordered carrier protein domains (3, 4). During polyketide, fatty acid, and nonribosomal peptide biosynthesis, the carrier protein serves as a scaffold to tether the building blocks and growing products as the component pieces are assembled and modified. The carrier protein must therefore interact with all enzyme domains responsible for loading carbon units, condensing these units, modifying the condensation product, and cleaving the final product from the synthase (Figure 1). Metabolic engineering, a major goal within the biosynthetic community, is premised upon the assumption that domains within the modular framework of these synthases may be stitched together to produce viable assembly lines, yet our understanding of basic interactions between these protein domains remains www.acschemicalbiology.org
incomplete. Progress has been hindered by the lack of structural data for many of these systems, and recent structures of these megasynthases contain substantial gaps (3–5). In such megasynthases, the acyl carrier protein (ACP) domain interacts with a variety of partner domains that can span great distances (up to ⬃100 Å). Sequence alignment and mutagenesis experiments have informed some features of carrier protein binding by these domains (6–8). Modular biosyntheses require the activity of condensing enzymes to extend the molecular backbone of their products; in fatty acid and polyketide synthesis, these enzymes are ketosynthases. Prior to catalyzing the condensation reaction, the active site cysteine of the extension ketosynthases (KASI and KASII of Escherichia coli fatty acid synthase) accepts the growing product chain from an upstream carrier protein. Thecarbon backbone is then extended by decarboxylative condensation of the downstream carrier protein-tethered malonate or methylmalonate. In fatty acid synthesis, each subsequent step in condensation only occurs after the -ketone of the growing chain bound to an upstream carrier protein becomes fully reduced by the stepwise activity of the ketoreductase, dehydratase, and enoyl reductase domains (Figure 1). Each of the enzymes in this pathway must recognize the identity of both the carrier protein and tethered substrate in order for
A B S T R A C T Fatty acid, polyketide, and nonribosomal peptide biosynthetic enzymes perform structural modifications upon small molecules that remain tethered to a carrier protein. This manuscript details the design and analysis of cross-linking substrates that are selective for acyl carrier proteins and their cognate condensing enzymes. These inactivators are engineered through a covalent linkage to fatty acid acyl carrier protein via post-translational modification to contain a reactive probe that traps the active site cysteine residue of ketosynthase domains. These proteomic tools are applied to Escherichia coli fatty acid synthase enzymes, where KASI and KASII selectively cross-link ACPbound epoxide and chloroacrylate moieties. These mechanism-based, protein–protein fusion reagents also demonstrated cross-linking of KASI to type II polyketide ACPs, while nonribosomal peptide carrier proteins showed no reactivity. Similar investigations into protein–protein interactions, proximity effects, and substrate specificities will be required to complete the mechanistic understanding of these pathways.
*Corresponding author,
[email protected]. Received for review September 15, 2006 and accepted October 19, 2006. Published online December 1, 2006 10.1021/cb6003965 CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. ACP partner domains in E. coli fatty acid synthesis. ACP is loaded with a malonyl moiety by malonyl-CoA:ACP transacylase (MAT). Fatty acid chain elongation at the ketoacyl-ACP synthase (KAS) produces an extended acyl chain bound to ACP, which is subsequently reduced to the saturated alkyl chain by the sequential action of -ketoacylACP reductase (KR), -hydroxyacyl-ACP dehydratase (DH), and enoyl-ACP reductase (ER). The fully reduced acyl-ACP then loads the ketosynthase for another round of chain extension, or the product is incorporated into other metabolic pathways.
accurate processivity to occur. Our initial studies focus on the E. coli fatty acid ketosynthase elongation enzymes, KASIII, which catalyzes the initial condensation reaction between acetyl-coenzyme A (CoA) and acylACP; KASI, which extends the fatty acid chain from C4 to C16; and KASII, which catalyzes the subsequent elongations. The crystal structure of each ketosynthase has been solved (9–11), but the interactions with ACP have been illustrated only from modeling studies of KASIII and ACP (12). Post-translational modification of carrier proteins via phosphopantetheinyltransferase (PPTase) is required for all modular synthase activity. We have previously demonstrated the selective use of CoA analogues to modify carrier proteins within in vitro and in vivo contexts (13, 14). To study the interaction between the ketosynthase domain and ACP, we synthesized pantetheine analogues containing terminal moieties that serve as irreversible cross-linking reagents. We began by modeling our target on the activity of cerulenin (1), a fungal metabolite well-known to serve as a mechanism-based inactivator of fatty acid and polyketide ketosynthase domains (Figure 2, panel a) (15–17). This inhibition occurs when the active site cysteine of the ketosynthase attacks the ␣-amidoepoxide moiety of ceru688
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lenin, forming an irreversible covalent adduct with the enzyme. We reasoned that a similar moiety could be covalently attached to a carrier protein domain via promiscuous CoA metabolic uptake and PPTasemediated carrier protein modification. This system would serve as a tool to probe protein–protein interactions between carrier proteins and ketosynthases. Utilizing our ability to attach almost any synthetic moiety to the terminus of a modified carrier protein, we could synthetically insert a mechanismbased inactivating functionality with selective distance from the modified serine residue of a carrier protein domain. We began with the design of a simple epoxide that could be installed through the addition of allylamine to pantothenic acid, or vitamin B5. The synthesis of the epoxy-pantetheine analogue 2 (Figure 2, panel a) was achieved by allylamine coupling with isopropylideneprotected pantothenic acid 3 using standard peptide coupling conditions, followed by acidic deprotection and epoxidation by dimethyldioxirane (DMDO) in acetone, to give 2 in an unoptimized 14% overall yield. Initial analysis of compound 2 indicated that the epoxide moiety hydrolyzed slowly under aqueous-buffered conditions. This led us to follow examples of rationally designed cysteine protease inhibitors; we also chose to design Michael acceptors appended to the pantetheine backbone as more stable probes of ketosynthase activity (18). Simple acrylamide pantetheine analogues were identified as potential thiol traps via 1,4conjugate addition. Here, we chose analogues that would exhibit slow reactivity with nonspecific nucleophiles and selective reactivity with activated cysteine nucleophiles. In an effort to eliminate retroaddition of the inactivated complex, -chloroacrylamide-containing pantetheine analogues 4 and 5 were chosen as targets for the ability to undergo tandem 1,4-conjugate addition and -chloro-elimination to yield irreversible complexes. The cis and trans analogues were synthesized from the previously WORTHINGTON ET AL.
described amine 6 (19) via coupling with chloroacrylate under standard conditions and acidic deprotection to give 4 and 5, each in an unoptimized yield of 23% for the two steps (Figure 2, panel b). The pantetheine analogues thus created were analyzed for activity with CoaA (PanK), the first enzyme in the CoA biosynthetic pathway that serves as the gatekeeper and carries out the rate-limiting step in the biosynthesis of CoA (20). Should compounds 2, 4, and 5 serve as acceptable substrates with CoaA, we can assume that reactions with the partner enzymes for attaching 4=-phosphopantetheine to carrier proteins will proceed smoothly in vitro (13). In a coupled assay that monitors the consumption of ATP, these substrates showed kcat/Km values comparable to that of pantetheine, but much lower than the natural substrate pantothenate (see Supplementary Table 1). These arise from similar turnover numbers (kcat) but elevated binding constants (Km) compared to those of pantothenate. Importantly, no enzyme inactivation by the electrophile-containing pantetheine analogues was detected. This established that 2, 4, and 5 should be acceptable substrates for in vitro conversion to CoA analogues and subsequent PPTase attachment onto carrier proteins. Pantetheine analogues 2, 4, and 5 were then coupled with purified E. coli fatty acid apo-ACP via a one-pot CoA enzymatic synthesis and PPTase transfer through the tandem activity of recombinant E. coli CoaA, CoaD, CoaE, and Bacillus subtilis Sfp (13, 21). To solutions of modified crypto-ACPs 7, 8, and 9, E. coli recombinant KASI, KASII, and KASIII were added (Figure 3). Where each enzyme performs acyltransferase activity with acyl-ACP, we anticipated the formation of a fusion complex through the mechanism-based inactivation of each ketosynthase from active site cysteine attack of the epoxide in 7 to give 10 or tandem Michael addition/-elimination with 8 or 9 to give 11 (Figure 3). KASIII performs its substrate loading, or acyltranswww.acschemicalbiology.org
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Figure 2. Pantetheine analogue synthesis. a) Synthesis of epoxide-functionalized pantetheine 2. Cerulenin 1 serves as our model for ketosynthase inactivation. Isopropylidene-protected pantothenic acid 3 was coupled to allylamine. Subsequent deprotection and epoxidation with DMDO afforded 2. b) Synthesis of -chloroacrylatepantetheine analogues 4 and 5. Compound 6 was coupled to cis- and trans-3chloroacrylic acid, followed by deprotection, to afford 4 and 5.
ferase, step with acetyl-CoA as the acyl donor, in contrast to KASI and KASII, which utilize carrier protein-bound moieties. Therefore, KASIII would not be expected to form a fusion construct with these tools. However, KASI and KASII, which perform their acyltransferase step with acyl-ACP, should be crosslinked to ACP. The resulting complexes KASI– ACP and KASII–ACP would therefore contain an irreversibly blocked ketosynthase activity, thus abrogating fatty acid processivity. The one-pot incubation of 2, 4, or 5 with the CoA biosynthetic enzymes, the PPTase Sfp, ACP, and either of the ketosynthases KASI or KASII generates irreversible covalent cross-linking between ACP and the ketosynthase domain (Figure 4). These products resulted in observed gel shifts of the ketowww.acschemicalbiology.org
synthase band from an observed mass ⬃50 kDa for the ketosynthase to ⬃80 kDa for the ACP–KAS complex when analyzed by SDS-PAGE (Figure 4, panel b). Extra bands seen in the regions of KASI and ACP–KAS of our negative control (Figure 4, panel b, lanes 1c and 2c) were contaminants that eluted with our His-tagged proteins during metal affinity chromatography. The large gel shift of the complex ACP–KAS would be expected, as the acidic ACP is known to run at an observed mass greater than ⬃20 kDa on SDS-PAGE (22). Nevertheless, the unusually large gel shift of the ketosynthase upon ACP binding merited further investigation, discussed below. SDS-PAGE analysis confirmed that no other enzymes in the mixture interacted with the modified crypto-ACP 7,
8, or 9. In addition, no interaction between modified ACP and KASIII was detected (data not shown). While each of the compounds gave the same cross-linking effect, the chloroacrylamide compounds 4 and 5 gave superior efficiency in cross-linking than the epoxide 2 in their interactions with both KASI and KASII. This is likely due to slow hydrolysis of the epoxide prior to interaction with the ketosynthase active site cysteine. However, it is also possible that the extended reactive centers of 4 and 5 favor ACP interaction. The site of nucleophilic attack for both 4 and 5 is two carbon lengths longer than the normal acylated pantetheine, while the terminal epoxide of 2 is one carbon length shorter. This chain length preference by the ketosynthase is currently being investigated. Nevertheless, these results suggest rather permissive acyl-ACP substrate loading by the E. coli fatty acid ketosynthases. In addition, there is a small but observable preference by both ketosynthases for the trans-isomer 4 over the ciscompound 5, as visualized by band intensity. This preference has been observed in previous studies of structurally related cysteine proteases (18). To investigate the unusually large gel shift of the ketosynthase band on SDS-PAGE, cross-linked proteins 10 and 11 were analyzed using in gel digestion followed with MALDI-TOF tandem mass spectrometry to verify the identity of each enzyme partner as well as the site-specificity of the crosslinking reaction. Considerable portions of both the ACP and each ketosynthase were detected (see Supplementary Figure 1). Importantly, active site residues Ser36 for ACP, Cys163 for KASI, and Cys164 for KASII were not identified in three independent analyses, suggesting the covalent modification had formed as designed in a mechanismbased manner. In addition, the lack of interaction between modified ACP and other enzymes in the one-pot reaction further supported the specific nature of the crosslinking between Ser36 of ACP and the active VOL.1 NO.11 • 687–691 • 2006
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Figure 3. The one-pot formation of carrier protein–ketosynthase (CP–KS) complexes in vitro. Pantetheine analogues 2, 4, or 5 are incubated with CoaA, CoaD, and CoaE in the presence of ATP to generate CoA derivatives. Apo-CP is modified by Sfp to incorporate the functionalized pantetheine arms of these derivatives into crypto-CPs 7, 8, and 9. Reaction of crypto-CP with the active site cysteine of a ketosynthase domain generates the covalent cross-link between carrier protein and ketosynthase.
site cysteine of KASI and KASII. Because of the nature of the proteomic analysis used, we were unable to observe a peptide fragment representative of the region incorporating both active sites. We have chosen to address this issue using protein crystallographic studies, which are forthcoming. Finally, the absence of reactivity between ACP and the conserved active site cysteine of KASIII suggests that the ACP binding motif of KASI and KASII is essential for substrate loading, while KASIII, which does bind ACP subsequent to priming by acetyl-CoA, may undergo a conformational change upon substrate loading. It is also probable that KASIII reacted with residual CoA analogues formed in situ within the one-pot reaction. Because KASIII utilizes acetyl-CoA as an acyl donor, the absence of cross-linking by KASIII with these carrier protein analogues indicates that the cross-linking activity may be used to visualize protein–protein interactions between enzymes that demonstrate partner reactivity (Figure 1). To establish this potential, we investigated the use of pantetheine analogue 4 for specificity of KASI with recombinant carrier protein domains active in natural product biosyntheses. These included two carrier proteins from nonribosomal peptide synthases, VibB (from Vibrio cholerae) and EntB (from E. coli), as well as two carrier proteins from type II polyketide synthases, FrenACP (from Streptomyces roseofulvus) and OtcACP 690
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(from Streptomyces rimosus) (Figure 4, panel c). Cross-linking with KASI was seen only with FrenACP and OtcACP, while VibB and EntB remained unmodified. Clear preference for E. coli ACP could be seen by comparison of intensity in the cross-linked bands. This result reflects the well-known sequence and activity-based homology between type II fatty acid ACPs and type II polyketide synthase ACPs. Indeed, type II polyketide ACPs are known to catalyze transformations with type II fatty acid synthase machinery from E. coli and streptomycete hosts (23–25). A lack of cross-linking between KASI and nonribosomal peptide carrier proteins indicates a distinct specificity in protein–protein interaction that is not satisfied with these pairings. The details of these interactions may be better understood through structural studies. Efforts to further understand the effect of specific residues upon protein–protein interactions in these modular synthases remain essential toward understanding their connected activity. Here, we have shown the utility of chemoenzymatic synthesis of post-translationally modified carrier proteins to generate a mechanism-based cross-linking reagent. This approach has the potential for general applicability to all systems involving carrier protein-mediated acyl transfer. These molecules may prove to be potent mechanismbased inactivators of type II fatty acid biosynthetic machinery in vivo. We also foresee WORTHINGTON ET AL.
the application of similar cross-linking experiments to probe substrate selectivity of ACP-mediated synthases via modified carrier proteins. METHODS Synthetic protocols, compound characterization, and additional experimental procedures may be found in the Supporting Information. Kinetics. Kinetic analysis was performed according to the protocol of Strauss and Begley (26). Cross-Linking of Recombinant ACP and Ketosynthase. To compare 2, 4, and 5 by SDSPAGE, we used the following procedure. To a 50 mM potassium phosphate, pH 7.0, buffer with 12.5 mM Mg2Cl2 and 8 mM ATP were added ACP (10 g, 5-fold excess), ketosynthase (10 g), CoaA (1 g), CoaD (1 g), CoaE (1 g), and B. subtilis Sfp (10 M). To the mixture, we added 2, 4, and 5 (2 mM), and the mixture was incubated at 37 °C for a minimum of 1 h. Negative controls contained no pantetheine analogue. Reaction times ranging from 1 h to overnight were tested for these reactions, and we found that a 1 h incubation time was sufficient. Samples were run on a 9% SDS-PAGE, where cross-linked product was detected by staining with Coomassie blue stain. To compare fatty acid, polyketide, and nonribosomal peptide carrier proteins with SDS-PAGE, we used the following procedure. To a 50 mM potassium phosphate, pH 7.0, buffer with 12.5 mM Mg2Cl2 and 8 mM ATP were added carrier protein (5–10 g, 3-fold excess), KASI (10 g), CoaA (0.1 g), CoaD (0.1 g), CoaE (0.1 g), and B. subtilis Sfp (10 M). To the mixture, we added 4 (4 mM), and the mixture was incubated at 37 °C for a minimum of 1 h. Samples were run on a 10% SDS-PAGE, where cross-linked product was detected by staining with Coomassie blue stain. Acknowledgment: Funding was provided by the University of California, San Diego, Department of Chemistry and Biochemistry, NIH RO1GM075797, and ACS PRF 42158-G4. H.R. was supported as an NIH NIGMS PREP scholar. We thank Betsy Komives, Jim La Clair, Joe Noel, and Mike Austin for helpful
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LETTER Supporting Information Available: This material is available free of charge via the Internet.
REFERENCES
Figure 4. Application of the method for CP–KAS formation to the E. coli fatty acid synthase pathway. a) Apo-ACP is incubated with CoaA, CoaD, CoaE, and Sfp in the presence of ATP and 2, 4, or 5 to generate crypto-ACP with a pantetheine (p) analogue sidearm. Crypto-ACP reacts with the active site cysteine of the ketosynthase to form the CP– KAS fusion product. b) SDS-PAGE analysis of one-pot reactions to form ACP–KAS complexes. ACP was reacted with KASI in lanes 1a– d and with KASII in lanes 2a– d. Lanes designated “a” were incubated with compound 5, “b” with 4, and “d” with 2; lanes “c” were negative controls with no pantetheine analogue. c) SDS-PAGE analysis of one-pot reactions to form CP–KASI complexes using compound 4. KASI was reacted against water (as a negative control), E. coli ACP, FrenACP, OtcACP, EntB, and VibB in lanes 1– 6, respectively. discussions. Additionally, we thank Jordan Meier for providing compound 6 and H. Mori from the Japanese E. coli consortium for providing genes from the ASKA plasmid ORF library (27).
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