Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Abstract

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.

Keywords: enzyme turnover; polyketide; protein chimera; protein engineering; protein-protein interaction.

FIGURE 1.

Architecture of DEBS and structure of the resulting product 6-deoxyerythronolide B (6-dEB, 1). The modular architecture of the three constituent proteins (DEBS1, DEBS2, and DEBS3) is shown in cartoon form, together with the product of each catalytic module attached to its acyl carrier protein (ACP) domain. AT, acyltransferase; KS, ketosynthase; KR, ketoreductase (KR0, inactive KR); DH, dehydratase; ER, enoylreductase; TE, thioesterase. Black tabs represent docking domains, short C- and N-terminal polypeptides that enable selective interactions between specific pairs of individual polypeptides.

FIGURE 2.

Catalytic cycle of a bimodular DEBS derivative. This mini assembly line is comprised of three proteins: the LDD (shown in red), DEBS module 1 (M1, shown in yellow), and DEBS module 2 fused to the TE domain (M2+TE, shown in blue/gray). The acyltransferase (AT) of the LDD specifically transfers the propionyl moiety of propionyl-CoA (step 1) to the terminal thiol of the phosphopantetheinylated ACP (step 2) of the LDD. This primer unit is then translocated by acylation of the active site Cys-SH of the KS domain of DEBS M1 (step 3). Meanwhile, the ACP of DEBS M1 is loaded with a methylmalonyl extender unit by the action of the acyltransferase domain of DEBS M1. KS-catalyzed chain elongation by decarboxylative Claisen condensation yields an ACP-bound β-ketoacyl-diketide intermediate (step 4). In DEBS M1, the KR domain then catalyzes an epimerization of the C-2 methyl group followed by diastereospecific reduction (step 5) to give the mature (2S,3R)-diketide, which is then translocated to DEBS M2 via a thioester to thiol transacylation. There it undergoes another round of chain elongation and KR-catalyzed reduction (without epimerization), followed by (TE)-catalyzed release and lactonization (steps 6 and 7).

FIGURE 3.

Chimeric bimodular PKSs. Each PKS included LDD(4) and DEBS (5)M1(2) in combination with (3)Module+TE as the variable acceptor. Acceptor modules were derived from DEBS, rifamycin synthase (RIFS), or rapamycin synthase (RAPS). Compatible docking domains from DEBS (depicted as black tabs or numbers in parentheses) were fused to the corresponding C- and N-terminal ends of the respective donor and acceptor modules to enhance the specificity and efficiency of intermodular chain translocation. The predicted triketide products 2–8 generated in the presence of propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA, in the case of RIFS M2+TE), and NADPH, are shown for each chimeric module pair. All acceptor modules are specific for methylmalonyl-CoA, except for RIFS M2, which prefers malonyl-CoA but can also accept methylmalonyl-CoA.

FIGURE 4.

Chimeric trimodular PKSs. Each PKS includes LDD(4), DEBS (5)M1(2), a variable (3)Module(2), and DEBS (3)M3+TE as the terminal module. The variable intervening modules were derived from DEBS, RIFS, or RAPS. Compatible docking domains from DEBS (depicted as black tabs or numbers in parentheses) were fused to the C and N termini of each module. The predicted triketide products 9–15, generated in the presence of propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA, in the case of RIFS M2+TE), and NADPH, are shown above each trimodular combination. All acceptor modules were specific for methylmalonyl-CoA except for RIFS M2, which prefers malonyl-CoA but can also accept methylmalonyl-CoA.

FIGURE 5.

Yields and purity of proteins used. A and B, SDS-PAGE analysis of individual PKS modules used to reconstitute chimeric bimodular (A) and trimodular (B) assembly lines are shown. C, SDS-PAGE analysis of mutant and hybrid modules used in this study is shown. The yield of each protein from a recombinant E. coli culture is summarized in the panel to the right.

FIGURE 6.

Turnover rates of chimeric bimodular (A) and trimodular (B) PKSs. All initial rate data were obtained at individual PKS protein concentrations of 4 μm and non-limiting concentrations of propionyl-CoA, methylmalonyl-CoA, and NADPH. In assays containing RIFS M2+TE, malonyl-CoA was also included because this module prefers malonyl extender units, although exclusion of malonyl-CoA did not affect the turnover rate of this system. Dashed lines indicate the threshold rate of NADPH consumption in the absence of the chimeric module. Error bars indicate averages of two measurements (each performed in triplicate) obtained with independent protein preparations. Numerical values for all bars are provided in supplemental Tables S6 and S7.

FIGURE 7.

LC-MS analysis of triketide products. A, lactones 2 and 4 (C₉H₁₆O₃, calculated molecular weight 172.110) were detected in reaction mixtures containing the reference module (DEBS (3)M2+TE), as well as DEBS (3)M5+TE and (3)M6+TE, and RIFS (3)M5+TE and (3)M7+TE. B, ketolactone 3 (C₉H₁₄O₃, calculated molecular weight 170.090) was detected in reaction mixtures containing DEBS (3)M3+TE and RIFS (3)M2+TE. Ketolactone 8 (C₈H₁₁O₃, calculated molecular weight 156.080) was detected in reaction mixtures containing RIFS (3)M2+TE with malonyl-CoA. For all systems the extracted ion chromatograms, obtained by extraction of the [M + Na]⁺ species, are shown, and the peak of interest is marked by an arrow based on its characteristic mass spectrum, shown explicitly in the case of DEBS (3)M2+TE. (Labeled peaks from left to right correspond to [M + H-H₂O]⁺, [M + H]⁺, and [M + Na]⁺ ions.)

FIGURE 8.

LC-MS analysis of tetraketide products. A, lactone 10 (C₁₂H₂₀O₄, calculated molecular weight 228.140) was detected in reaction mixtures containing the reference module (DEBS (3)M2(2)), as well as RIFS (3)M5(2). B, pyrone 9 (C₁₂H₁₈O₄, calculated molecular weight 226.120) was detected in reaction mixtures containing RIFS (3)M2(2) and (3)M3(2), as well as RAPS (3)M6(2). For all systems the extracted ion chromatograms, obtained by extraction of the [M + Na]⁺ species, are shown, and the peak of interest is marked by an arrow based on its characteristic mass spectrum, shown explicitly in the case of DEBS (3)M2(2). (Labeled peaks from left to right correspond to [M + H-H₂O]⁺, [M + H]⁺, and [M + Na]⁺ ions.)

FIGURE 9.

Influence of substrate-KS recognition on chimeric PKSs. A, chimeric bimodular PKSs harboring the DEBS M1-KR2 mutant. Each PKS included LDD(4) and DEBS (5)M1-KR2(2) in combination with (3)Module+TE as the variable acceptor. The predicted triketide products 16–18 generated in the presence of propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA, in the case of RIFS M2+TE), and NADPH, are shown for each chimeric module pair. B, turnover rates of bimodular constructs consisting of DEBS LDD, DEBS M1 plus the designated acceptor module (black bars) and turnover rates of the same systems with DEBS M1-KR2 mutant (gray bars) in place of DEBS M1. All initial rate data were obtained at individual PKS protein concentrations of 4 μm and non-limiting concentrations of propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA, in the case of RIFS M2+TE), and NADPH. The dashed line indicates the threshold rate of NADPH consumption in the absence of the chimeric module. Error bars indicate averages of three measurements. Numerical data from this bar graph are summarized in supplemental Table S8.

FIGURE 10.

LC-MS analysis of diastereomeric triketide lactones produced by bimodular chimeric PKS harboring either DEBS M1 (left) or DEBS M1-KR2 (right). Diastereomeric triketide lactones 2 and 17 (C₉H₁₆O₃, calculated molecular weight 172.110) were detected in reaction mixtures containing DEBS (3)M2+TE, as well as DEBS (3)M5+TE and (3)M6+TE. A reference sample of lactone 17 was produced by self-priming of DEBS3 with methylmalonyl-CoA (28); the two diastereomers are distinguishable by LC-MS analysis (supplemental Fig. S1). Diastereomeric triketide lactones 3 and 16 (C₉H₁₄O₃, calculated molecular weight 170.090) were detected in reaction mixtures containing DEBS (3)M3+TE and RAPS (3)M6+TE. In all cases the extracted ion chromatograms corresponding to the [M + Na]⁺ species are shown, and the peak of interest is marked by an arrow.