Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Abstract

Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.assembly line enzymes | polyketide synthase | protein-protein interactions M odular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) are assembly lines of catalytic modules responsible for the biosynthesis of polyketide natural products (1-6). A fundamental challenge toward elucidating their molecular logic is to understand the mechanism by which covalently bound biosynthetic intermediates are sequentially accessed by individual catalytic sites of the synthase. Most notably, each module of a polyketide synthase (PKS) has an acyl carrier protein (ACP) domain that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze polyketide chain elongation, and subsequently engages with the KS domain of the next module to facilitate forward chain transfer (Fig. 1). The simplest explanation for the orderly progress of a growing polyketide chain along a multimodular PKS is that each ACP is equivalently recognized by both KS partners, and that selection of the appropriate KS-ACP pair at a given point in the catalytic cycle is solely dictated by the identity of the substrate anchored on the ACP. (In the chain elongation reaction, a nucleophilic malonyl extender unit is attached to the ACP, whereas the growing polyketide chain itself is attached to the ACP via an electrophilic thioester linkage when it is ready for forward transfer.) Previous studies, however, suggest that protein-protein recognition between the KS and the ACP domains also plays an important role in both reactions (7-10). Moreover, this protein-protein recognition not only influences the specificity (k cat ∕K M ) of each reaction, but also the maximum rate constant (k cat ).In the course of our efforts to elucidate the principles that govern KS-ACP recognition during chain elongation and chain transfer, we have made two unexpected and potentially important observations. First, notwithstanding the fact that the same active sites are deployed in both reactions, the structural elements that mediate KS-ACP recognition are entir...

Probing the interactions of an acyl carrier protein domain from the 6‐deoxyerythronolide B synthase

“…8 Remarkably, even though both KS partners of a given ACP are homologous, KS-ACP recognition appears to be controlled by distinct types of encounters. [8][9][10] Although intermodular chain translocation is controlled by helix 1 of the ACP (corresponding to P13-G33 in ACP2), chain elongation is controlled by loop 1 (H34-D52 in ACP2). In principle at least, this orthogonal control strategy provides a conceptual model to explain the mechanistic logic of assemblyline biosynthesis.…”

“…This is entirely consistent with recent protein engineering experiments that established that helix 1 (corresponding to P13-G33 in ACP2) plays a critical role in intermodular chain translocation. 10 Disappearance of T68 and L72 not only suggests that loop 2 and the C-terminus of helix 2 of the ACP are affected by these protein-protein interactions but also highlights the complementary analytical potential of NMR spectroscopy. Because these residues are highly conserved among the ACP domains of multimodular PKSs, they would not have been identified in earlier experiments that were based on the construction and analysis of chimeric ACPs.…”

“…Because these residues are highly conserved among the ACP domains of multimodular PKSs, they would not have been identified in earlier experiments that were based on the construction and analysis of chimeric ACPs. 10 Several other peaks display significant loss of intensity (>60%, 2 standard deviations ¼ 30%) upon titration of [KS3][AT3] into a solution of 15 N-labeled ACP2 [ Fig. 4(B)].…”

“…5) accounts for the biophysical data presented here and is also in agreement with the previously published biochemical findings. 10,16 According to this model, the ACP2 domain docks in the deep cleft of [KS3][AT3] that is established by the KS-AT interdomain linker region. The binding sites on helix 1 of ACP2 are within 4 Å of the AT3 domain.…”

“…8,9 Remarkably, they do so via protein-protein interactions that are energetically orthogonal during chain elongation to those that are important to chain translocation. 10 To derive structural evidence for the existence of selective protein-protein interactions involving ACP domains, we performed a series of NMR experiments using the structurally characterized ACP domain from module 2 of DEBS ( Fig. 2) 11 and two structurally characterized [KS][AT] didomains from modules 3 and 5 of DEBS.…”