Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase

Abstract

Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.

Figure 1

Modular organization of 6-deoxyerythronolide B synthase (DEBS). Each catalytic domain is represented by a block diagram. KS: ketosynthase; AT: acyl transferase; ACP: acyl carrier protein; DH: dehydratase; ER: enoyl reductase; KR: ketoreductase; and TE: thioesterase. KR⁰ denotes an inactive ketoreductase domain. The phosphopantetheine prosthetic group of ACP is drawn as a curly line. Inter-protein linkers are shown as matching tabs.

Figure 2

Chain elongation cycle catalyzed by DEBS module 3 + TE (M3TE). AT is acylated with a methylmalonyl extender unit from its CoA-derivative, which gets transferred to the downstream ACP. KS is primed with a 2-methyl-3-hydroxy pentanoyl unit from diketide compound 1. Condensation takes place in the active site of KS with the release of carbon dioxide. TE catalyzes lactone formation with release of the final product.

Figure 3

The deconstructed module system: chain elongation performed by [KS][AT] didomain and standalone ACP. Non-cognate nucleophile can also be introduced via acyl phosphopantetheinylation of apo-ACPs catalyzed by Sfp enzyme.

Figure 4

The hybrid module containing [KS3][AT3] and [KR5][ACP5][TE].

Figure 5

Proteins used in the assays (left to right): [KS3][AT3], [KS6][AT6], ACP1(0), ACP2(0), ACP3(0), ACP4(0), ACP5(0), ACP6(0), ACP2(2), ACP3(2), ACP4(4), M3TE, and M3M5TE.

Figure 6

(A) [KS3][AT3]-and (B) [KS6][AT6]-catalyzed chain elongation velocity as a function of ACP concentration for each of the six ACP domains from DEBS.

Figure 7

[KS3][AT3]-catalyzed chain elongation velocity as a function of ACP concentration for two forms of (A) ACP2, (B) ACP3, and (C) ACP4. Solid line: ACP without C-terminal linker; dashed line: ACP with C-terminal linker.

Figure 8

(A) Radio SDS-PAGE: [¹⁴C]Compound 1 labeling of hybrid module M3M5TE and ACP2(2). (B) Radio SDS-PAGE: [¹⁴C]Methylmalonyl-CoA labeling of M3M5TE and ACP5. (C) Radio TLC: Triketide lactone formation by M3M5TE. Lane 1, positive control reaction (module 3+TE, [¹⁴C]methylmalonyl-CoA, compound 1); lane 2, chain elongation by M3M5TE ([¹⁴C]methylmalonyl-CoA, compound 1); lane 3, chain elongation and reduction by M3M5TE ([¹⁴C]methylmalonyl-CoA, compound 1, NADPH).