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.

Fig. 1.

Chain elongation cycle catalyzed by a PKS module. Each module in a PKS consists of a unique set of covalently linked catalytic domains responsible for a single round of chain elongation via the following steps. (1) KS is primed with a growing polyketide chain by the upstream ACP (grayscale). AT is acylated with a CoA derived methylmalonyl extender unit (2), which is transferred to the downstream ACP (black and white) (3) and a decarboxylative condensation takes place in the active site of KS (4). The ACP tethered extended polyketide chain is processed by the tailoring domains (for example KR, process not shown) and subsequently transferred to the downstream KS domain as in (1). The phosphopantetheine prosthetic group of the ACP is drawn as a wavy line. Interprotein linkers that are known to facilitate intermodular chain transfer are shown as matching solid tabs (black). KS, β-ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; KR, ketoreductase.

Fig. 2.

Structure-based hybrid ACP construction and analysis. (A) Solution structure of the ACP domain from DEBS module 2. To recombine the predicted secondary structure elements of ACP3 and ACP6 (helices H_I, H_II, and H_III, and loops L_I and L_II), four fusion sites marked by black arrows were identified. (B) Sequence alignment of DEBS ACP2, ACP3, ACP6, and a representative chimeric protein [construct SHIV25, whose model is shown in (C)]. In SHIV25 the N-terminal sequence of ACP6 is fused to the C-terminus of ACP3 at the loop I—helix I junction. The asterisk indicates the conserved phosphopantetheine attachment site Ser54. The arrows mark the fusion sites. Other fusion sites used to generate chimeras of ACP3 and ACP6 detailed in Fig. 3 are indicated by residue number. Red: ACP3-derived. Green: ACP6-derived. (D) Chain elongation activity of ACP3, ACP6, and the chimeric ACP SHIV25. Each ACP was assayed separately with [KS3][AT3] and [KS6][AT6] under conditions described in Materials and Methods section. The reaction product was visualized and quantified by radio-TLC.

Fig. 3.

Evaluation of chimeric ACP proteins in an assay for polyketide chain elongation. A series of chimeric ACP proteins were constructed and assayed with homodimeric [KS][AT] proteins harboring either KS3 or KS6. For each chimera the activity with [KS3][AT3] or [KS6][AT6] is normalized to the corresponding wild-type ACP. The initial set of chimeras are shown in panels A and B. (A) ACPs that prefer [KS3][AT3] over [KS][AT6]. (B) ACPs that prefer [KS6][AT6] over [KS3][AT3]. Because these preferences tracked with the identity of L_I, SHIV22 (C) and AYC79 (D) were constructed and found to have the predicted [KS][AT] preference, albeit at the expense of reduced activity. Additional chimeras, shown in Panels E and F, were engineered to further optimize activity and specificity. Specifically, redefinition of the L_I-H_II junction of SHIV22 and AYC79 afforded SHIV24 (E) and SHIV29 (F), respectively. The color scheme is similar to Fig. 2 (red = ACP3 derived, green = ACP6 derived). Fusion sites (black bars) are consistent with Fig. 2. For SHIV24 and SHIV29 the C-terminus of the substituted fragment is at residue 58. SHIV22, 25, 26 and 29 harbor a H26A mutation (see SI Text for details).

Fig. 4.

Docking model for ACP5 domain with the homodimeric [KS5][AT5] protein. Although the ACP domain of monomer A (gray) occupies a deep cleft between the KS and AT domains of monomer A (light cyan) (A), the conserved serine (red sticks) (B) at the N-terminal end of H_II is positioned to participate in polyketide chain elongation with the KS active site of monomer B (light green). The KS-AT linker region of each monomer, which interacts with L_I of the ACP domain (gray), is highlighted (dark cyan or dark green). The inset shows the two regions that show electrostatic complementarity with residue 44 (region I) and residue 45 (region II).

Fig. 5.

Evaluation of chimeric ACP proteins in an assay for intermodular transfer of a growing polyketide chain. Chimeras of ACP2 and ACP4 were tested for their ability to accept a growing polyketide chain from KS3, the microscopic reverse of the normal intermodular chain transfer event, which is specific for ACP2 (8). The color scheme is: red = ACP2 derived, green = ACP4 derived. The rate of chain transfer for each chimera is normalized to corresponding value for the wild-type ACP2. “L2” refers to the C-terminal sequence of ACP2 that docks onto an N-terminal coiled-coil on module 3, and is essential for efficient intermodular chain transfer (35). The junctions utilized for constructing chimeras of ACP2 and ACP4 are defined in Fig. S8. Fusion sites distinct from the ones indicated by black arrows in Fig. S8 are color coded, and are defined by residue number consistent with the numbering in Fig. S8.

Fig. 6.

Distinct interfaces mediate protein–protein interactions. Chain elongation (orange with residues 44 and 45 shown in red) and chain transfer (blue) epitopes lie on entirely different faces of the ACP domain.