Polyketide β-branching in bryostatin biosynthesis: identification of surrogate acetyl-ACP donors for BryR, an HMG-ACP synthase

Abstract

In vitro analysis of natural product biosynthetic gene products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in β-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE, and Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.

Figure 1

Portions of the pathway utilized in β-branching are highlighted with color in this depiction of the bryostatin biosynthetic pathway. BryC, BryX and BryD are not shown [1]. Though the complete structure of the PKC modulator, bryostatin 1 is shown here, the full suite of bryostatin molecules contains acyl chain variability at both C7 and C20. ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; FkbH, homolog to FkbH [38]; HMGS, HMG-CoA synthase homolog; KR, ketoreductase; KS, ketosynthase; KS_DC, decarboxylative ketosynthase; MT, methyltransferase; Unk, domain with unknown function. Refer to Figure S1 for more information.

Figure 2

Proteins and/or domains involved in HMG generation. A) HMG generation in the mevalonate pathway and B) during polyketide β-branching in PKS and mixed biosynthetic pathways. The covalent, enzyme-bound intermediate from the reaction being analyzed in this paper is boxed. C) The HMGS cassette ACP_D subclass of acyl carrier proteins can be observed in the phylogenetic tree generated using Jalview software (average distance BIOSUM2)[39]. Refer to Figure S2 for more information.

Figure 3

BryR catalyzed generation of HMG-BryM3 ACP from Ac-MacpC. A) As monitored by FTICR-MS, data are presented as m/z versus abundance. PPant ejection assay data from the entire charge state distribution are presented. PPant ejection peaks are in the +1 charge state. Intact donor and acceptor ACP data are also illustrated. Holo- and Ac-MacpC peaks are shown in the +13 charge state. Acac- and HMG-BryM3 are shown in the +14 charge state. Particular charge states illustrated are representative of the entire charge state envelope. B) Rado-TLC monitored acetyl transfer from [1-¹⁴C]-Ac-CurB to Acac-BryM3 ACP to form HMG-BryM3 ACP monitored by radio-SDS PAGE. Refer to Figures S4–9 for more information.

Figure 4

Binding of apo-ACPs to immobilized BryR, monitored by SPR. A) Each data point is the average of triplicate measurements; error bars are standard deviation. The data were fit to a one-site binding model (Y = B_max*X/(K_D + X)). Y = fraction bound, B_max = maximal response, X = ACP concentration. Dissociation constants (K_D^s) are reported in B. Forms of ACPs shaded grey in the table were not tested. Refer to Figures S10–S11 for more information.