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, 140 (21), 6518-6521

Structure-Function Analysis of the Extended Conformation of a Polyketide Synthase Module

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Structure-Function Analysis of the Extended Conformation of a Polyketide Synthase Module

Xiuyuan Li et al. J Am Chem Soc.

Abstract

Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations-an "extended" architecture and an "arch-shaped" architecture-although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (Fab) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) Bimodular and (B) trimodular derivatives of DEBS. Domains are shown as spheres. KS = ketosynthase; AT = acyl transferase; ACP = acyl carrier protein; L = KS-AT linker domain; KR = ketoreductase; KR0 = redox-inactive, epimerase-active KR homologue; TE = thioesterase. Blue tabs depict docking domains that facilitate non-covalent association of successive modules. Module 2 domains are colored for ease of comparison with Figure 2.
Figure 2.
Figure 2.
Alternative conformational models of homodimeric PKS modules: (A) SAXS model of DEBS module 3 + TE; (B) cryo-EM model of module 5 of the pikromycin synthase. Docking domain (“coil”), blue; KS, green; KS-AT linker, cyan; AT, pink; KR, yellow; ACP, orange; TE, brown. The collinear domain organization is as shown in Figure 1. The two conformations differ primarily in the orientation of the AT and L domains relative to the KS.
Figure 3.
Figure 3.
Catalytic cycle of DEBS module 2. Domains are colored as in Figure 1. The cycle includes four reactions: translocation of the growing polyketide chain (I) into and (IV) out of the module, (II) transacylation of an extender unit from methylmalonyl-CoA to its ACP domain, and (III) chain elongation via decarboxylative C–C bond formation. Module 2 also reduces the β-keto thioester product of chain elongation; this reaction is lumped into step III.
Figure 4.
Figure 4.
Binding of 1B2 to the KS-AT fragment of DEBS module 3. (A) ELISA of binding of 1B2 with module 3 + TE. (B) SEC traces of 1B2 (blue), the KS-AT fragment (red), and the 1B2/KS-AT complex (green). (C) SDS-PAGE: (left lane) the major SEC peak is a stoichiometric mixture of 1B2 and KS-AT; (right lane) 1B2 alone.
Figure 5.
Figure 5.
Crystal structure of 1B2 in complex with the KS-AT fragment of DEBS module 3 + TE. Two views of the complex (PDB entry 6C9U) are shown. The coloring scheme is identical to that in Figures 1–3 (docking domain, blue; KS, green; KS-AT linker, cyan; AT, pink). This homodimer binds to two copies of the Fab, shown in red. The axis and plane of symmetry are shown in the lower view.
Figure 6.
Figure 6.
Effect of 1B2 binding on chain translocation and chain elongation by DEBS module 2 + TE. 1B2 binds tightly to DEBS module 2 + TE. The initial slope of the graph in (A) reflects conditions where chain translocation is rate-limiting, whereas the maximum velocities in (A) and (B) reflect conditions where elongation is rate-limiting. Blue curves show the activity without 1B2; red curves show the activity in the presence of 1.2 equiv of 1B2.
Figure 7.
Figure 7.
Tandem (SEC−SAXS) analysis of the 1B2/module 2 complex. (A) Docking models of 1B2 bound to each conformation from Figure 2 were generated by aligning the X-ray structure of the 1B2/KSAT complex (Figure 5) with each module conformation. The Fab complexed to the extended conformation is shown at the top, whereas the arched conformation is shown below. Domain coloring is the same as in Figures 2 and 5. (B) Comparisons of (left) log(I) vs q and (right) Kratky plots generated by SEC−SAXS analysis of the 1B2/module 2 complex (black curve) and CRYSOL analysis of the extended (blue curve) and arched (red curve) models from (A). (C) Comparison of (left) log(I) vs q and (right) Kratky plots from SEC−SAXS analysis of 1B2/module 2 trapped in states A (black curve), B (green curve), and D (purple curve). (cf. Figure 3).

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