β-Branching in the biosynthesis of bongkrekic acid: a complex affair

Abstract

Bongkrekic acid is a potent respiratory toxin which inhibits the mitochondrial ATP/ADP carrier protein. The polyketide synthase that biosynthesises bongkrekic acid recruits a discrete cassette of β-branching enzymes (BonF-BonI) to install two distinct β-branches: an endo-β-methyl branch in module 1, and a carboxymethyl β-branch in module 11. Both β-branches contribute to specific interactions with bongkrekic acid's biological target. However, a critical component of the β-branching cassette, the donor acyl carrier protein (ACP_D), has not been identified in previous studies. Furthermore for the module 11 carboxymethyl β-branch to be retained, conversion to an endo-β-methyl branch via the enoyl-coenzyme A hydratase (ECH), BonI, must be avoided. The mechanistic basis for these divergent β-branching pathways is poorly understood, both in the bongkrekic acid biosynthetic pathway and more generally where it arises in polyketide biosynthesis. Here, we confirm the roles of BonF-BonI by reconstituting β-branching in modules 1 and 11 in vitro and uncover the previously unannotated ACP_D, BonN, to complete the β-branching cassette. We further demonstrate promiscuous BonI interactions with both module 1 and 11 ACPs that confounds simple ACP selectivity arguments for carboxymethyl β-branch versus endo-β-methyl branch installation, suggesting that this is instead regulated by a complex interplay between substrate and kinetic control.

Fig. 1. Overview of bongkrekic acid biosynthesis. (A) Burkholderia gladioli pv. cocovenenans bon biosynthetic gene cluster (BGC). Genes are highlighted as follows: regulatory (grey), enoyl reductase (red), acyl hydrolase (green), acyltransferase (dark blue), β-branching cassette enzymes (orange), type I polyketide synthase (PKS, yellow), O-methyltransferase (OMT, light blue) and cytochrome P450 (CYP, purple). The newly discovered BonN is marked with an asterisk (this study). (B) Bongkrekic acid biosynthetic pathway. Type I PKS BonA and BonD incorporate β-branches at acceptor ACPs (ACP_As, blue arrows). β-Branches are shown in red. (C) Proposed divergent branch formation between modules BonA (i) and BonD (ii) to incorporate an endo-β-methyl branch and a carboxymethyl β-branch (both highlighted in red). In the context of an advanced polyketide intermediate, the carboxymethyl β-branch is equivalent to an MG-intermediate. The stereochemistry of the carboxylated β-branched intermediate is shown cis to the alkene proton of the β-ketothioester, with the polyketide intermediate trans. A similar bond rotation upon decarboxylation to form the endo β-branch is assumed as shown by Walker et al. (inset) HMGS cassette components.

Fig. 2. BonF-catalysed decarboxylation of malonyl-BonN. (A) Proposed reaction scheme for BonF-catalysed decarboxylation of malonyl-BonN. (B) Deconvoluted spectrum and corresponding Ppant ejection of BonF assay with malonyl-BonN. * refers to phosphogluconoylation of the His6 tag (+178 Da). (C) Scheme for BonF-catalysed malonyl-GbnF decarboxylation. (D) Deconvoluted spectrum and corresponding Ppant ejection of BonF assay with malonyl-GbnF. (E) Expected Ppant ejection ions.

Fig. 3. BonG-catalysed aldol addition scheme and ESMS assays. (A) Proposed reaction scheme for BonG-catalysed aldol addition of acetoacetyl (Acac)-BonD_ACP3b with acetyl (Ac)-BonN. (B) Deconvoluted spectrum and corresponding Ppant ejection of BonG assay with Acac-BonD_ACP3b and (Ac)-BonN. (Inset) Expected Ppant ejection ion for the HMG species.

Fig. 4. BonH- and BonI-catalysed dehydration and decarboxylation of HMG-BonA_ACP2a. (A) Reaction scheme to form endo-β-methyl-ACP2a from HMG-ACP2a via BonH and BonI, respectively. (B) Deconvoluted spectrum and corresponding Ppant ejection of HMG-ACP2a assays with BonH and BonI. (Inset) Expected Ppant ejection ion for HMG, MG and endo-β-methyl species.

Fig. 5. BonH- and BonI-catalysed dehydration and decarboxylation of HMG-BonD_ACP3b. (A) Reaction scheme whereby BonI converts MG-ACP3b to endo-β-methyl-ACP3b instead of retaining the carboxylated β-branch as observed in the biosynthetic pathway. (B) Deconvoluted spectrum and corresponding Ppant ejection of HMG-ACP3b assays with BonH and BonI. (Inset) Expected Ppant ejection ion for HMG, MG and endo-β-methyl species.

Fig. 6. Proposed alternate pathways that may act in the final stages of bongkrekic acid biosynthesis to retain the carboxymethyl β-branch prior to substrate off-loading. After aldol addition and dehydration of the polyketide attached to BonD_ACP3a/b (red). Substrate off-loading (blue) could occur via kinetic control by the terminal [KS] domain (pathway A) or steric occlusion of BonI by the [KS] domain (pathway B), prior to off-loading. The trans-acting BonM and BonL furnish bongkrekic acid/iso-bongkrekic acid.