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. 2012 Mar 13;109(11):4110-5.
doi: 10.1073/pnas.1118734109. Epub 2012 Feb 27.

Reprogramming a Module of the 6-deoxyerythronolide B Synthase for Iterative Chain Elongation

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Free PMC article

Reprogramming a Module of the 6-deoxyerythronolide B Synthase for Iterative Chain Elongation

Shiven Kapur et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Modular organization of the 6-deoxyerythronolide B synthase. Organized into three homodimeric polypeptides (DEBS1–3), DEBS consists of six modules, each containing a unique set of covalently linked domains. Noncovalent interactions localized to the termini of each polypeptide (matching black tabs) play an important role in chain translocation between modules 2 and 3 and modules 4 and 5. Together, the six modules of this assembly line utilize one propionyl-CoA-derived primer and six methylmalonyl-CoA-derived extender units to synthesize 6-deoxyerythronolide B (6-dEB). DH, dehydratase; ER, enoylreductase; TE, thioesterase; LDD, loading didomain. KR° in module 3 is inactive. The phosphopantetheine prosthetic group of the ACP is shown as a wavy line.
Fig. 2.
Fig. 2.
Catalytic cycle of a representative minimal module. Every module of an assembly line PKS catalyzes the following sequence of reactions: (1) the polyketide chain is translocated from the ACP domain of the preceding module (grayscale) onto the KS domain of the target module. (2 and 3) Separately, the AT domain catalyzes transfer of a selected extender unit (in this case, a methylmalonyl group) onto the ACP domain. (4) The KS domain then catalyzes chain elongation via decarboxylative condensation, leading to the formation of an ACP-tethered β-ketoacyl product. The oxidation state and stereochemistry of this product is then set by an appropriate combination of KR, dehydratase, and enoylreductase domains (not shown). Eventually, the chain is translocated to the downstream KS as in 1, although it is never translocated back to its own KS.
Fig. 3.
Fig. 3.
Identification of residues that contribute to KS–ACP specificity during chain elongation. (A) Schematic secondary structure of a typical ACP three-helix bundle. The approximate location (S) of the Ser residue onto which the phosphopantetheine arm is attached is shown. The secondary structure elements are mapped onto the tertiary structure in the ACP3 cartoon shown in B. Also shown is a multiple sequence alignment of ACP3, SHIV24, SHIV29, and ACP6 discussed in B. (B) An example of orthogonal KS–ACP recognition between modules 3 and 6 of DEBS. Also shown are two chimeric derivatives in which substitution of loop I of the ACP led to reversal of its preference for a KS partner. (C) Representative radio-thin-layer chromatography assay to quantify the relative preference of a given ACP for alternative KS partners. [KS3][AT3] and [KS6][AT6] refer to the ca. 190-kD homodimeric fragments of modules 3 and 6, respectively, that harbor both the KS and AT domains. (D) Site-directed mutagenesis of AYC79, a minimally altered chimeric derivative of ACP3 with greater preference for [KS6][AT6] than [KS3][AT3]. For the chain elongation activity assay, see SI Materials and Methods. Red, ACP3-derived sequences; green, ACP6-derived sequences. For steric reasons, an H26A mutation was necessary to obtain soluble protein (5).
Fig. 4.
Fig. 4.
Identification of residues that contribute to KS–ACP specificity during intermodular chain translocation. (A) KS–ACP specificity during chain translocation between DEBS modules 2 and 3. Whereas ACP2 and KS3 are effective partners, ACP4 cannot substitute for ACP2 in this reaction. However, a chimeric derivative of ACP4 (SHIV64), in which the first 10 residues of HI were replaced with their counterparts from ACP2, led to substantial improvement of its ability to partner with KS3 for chain translocation. (B) Site-directed mutagenesis of SHIV64. Chain translocation activity of each mutant is normalized to that of ACP4. For the activity assay, see SI Materials and Methods. Red, ACP2-derived sequences; green, ACP4-derived sequences. All constructs contain the C-terminal sequence of ACP2 that docks onto an N-terminal coiled-coil on module 3, and is essential for efficient intermodular chain translocation (6).
Fig. 5.
Fig. 5.
Models for KS–ACP interaction during chain elongation and chain translocation. (A) Docking of ACP5 on the structurally characterized [KS5][AT5] during chain elongation. (B) Docking of ACP4 on the structurally characterized [KS5][AT5] during chain translocation. In both images, the phosphopantetheine attachment site of the ACP is shown in black sticks; it interacts with the KS active site in the light green monomer. (C) Superposition of the two models shown in A and B. Residues 44 and 45 (red) at the chain elongation interface (orange) and residue 23 (deep blue) at the chain translocation interface (blue) are the principal mediators of KS–ACP recognition. The KS–AT linker region of each monomer in AC is highlighted (KSA-ATA, dark cyan; KSB-ATB dark green).
Fig. 6.
Fig. 6.
A ratchet model for unidirectional translocation of the growing polyketide chain. Sequence alignment of the N terminus of helix (HI) of adjacent ACP domains from (A) DEBS, (B) MEGS (the megalomicin PKS), and (C) TYLS (the tylosin PKS). Residues on this helix that are predicted to be partially or completely solvent exposed are underlined. Residue 23 (DEBS ACP2 numbering) is highlighted with an asterisk, and those that are 1 or 2 helical turns above or below this residue (i.e., residues 15, 19, and 26) are identified. The final ACP from each PKS is omitted because it does not have a downstream KS. (D) A ratchet model for unidirectional chain translocation. The [KS][AT] core of each module is compatible with the N-terminal sequence of helix I of the upstream ACP, but is mismatched to the homologous site on the ACP from its own module. The specificity of protein–protein interactions assures unidirectional translocation of ACP-bound intermediates. LDD, loading didomain.
Fig. 7.
Fig. 7.
An engineered chimeric ACP exhibits iterative chain elongation. (A) Schematic representation of the reactions catalyzed by [KS3][AT3] with the wild-type ACP3 (23) and the engineered ACP3 (SHIV78, shown as ACP3*). The reaction involving wild-type ACP3 follows the solid arrows leading to the formation of compound 1 (a triketide ketolactone). The reaction involving ACP3* is predicted to follow the dashed arrow, leading to the formation of compound 2 (a tetraketide ketolactone). (B) Radio thin-layer chromatography for the reaction of [KS3][AT3] with wild-type ACP3 (lane a) and ACP3* (lane b). Compounds 1 and 2 are labeled, and have identical Rf values to those of authentic standards (31) (C) The identity of the tetraketide was confirmed by an established proteolytic LC-high-resolution mass spectroscopy (HRMS) method (24). The HRMS trace (integrated from 60–63 min of the elution gradient) for the ACP3* reaction is shown. The ACP fragment shown in the panel (peptide sequence AFSELGLDS*LNAMALR, where the phosphopantetheine moiety containing the tetraketide thioester is covalently bound to the active serine, S*) has a calculated monoisotopic m/z of 1,138.551 (z = +2). Additional fragment isotopes are labeled and follow the expected isotopic distribution. The precision of the HRMS device is 2 millimass unit.

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