Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase

Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E.; Khosla, Chaitan

doi:10.1021/acs.biochem.6b00639

Cited by 62 publications

(63 citation statements)

References 19 publications

(76 reference statements)

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“…Chemie Zuschriften E. coli FabB also resulted in moderate to very low turnover of the substrate. [17,18] Ad ouble mutant (H3364A and H3404A) stalled the formation of 5,thus proving that these residues are indispensable for the Michael addition. According to functional studies with homologous KS domains, [17][18][19] H3404 activates the malonyl carboxylate and stabilizes the enolate.…”

Section: Methodsmentioning

confidence: 94%

“…[17,18] Ad ouble mutant (H3364A and H3404A) stalled the formation of 5,thus proving that these residues are indispensable for the Michael addition. According to functional studies with homologous KS domains, [17][18][19] H3404 activates the malonyl carboxylate and stabilizes the enolate. Reduction of the rates in the carbon-carbon bond formation as well as lactonization ( Figure 2C and 3A,v )i ndicates that H3404 plays am ajor role in all steps of the reaction.…”

Section: Methodsmentioning

confidence: 94%

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On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

et al. 2018

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Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In stark contrast, the KS of the rhizoxin PKS branching module mediates a Michael addition, which sets the basis for a pharmacophoric δ-lactone moiety. The precise role of the KS was evaluated by site-directed mutagenesis, chemical probes, and biotransformations. Biochemical and kinetic analyses helped to dissect branching and lactonization reactions and unequivocally assign the entire sequence to the KS. Probing the range of accepted substrates with diverse synthetic surrogates in vitro, we found that the KS tolerates defined acyl chain lengths to produce five- to seven-membered lactones. These results show that the KS is multifunctional, as it catalyzes β-branching and lactonization. Information on the increased product portfolio of the unusual, TE-independent on-line cyclization is relevant for synthetic biology approaches.

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Section: Methodsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 94%

On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

et al. 2018

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“…Despite these differences, a S315A mutation in loop 2 of DEBS KS1 increased the formation of propionate, which results from the decarboxylation of the methylmalonyl-ACP substrate. 46 These findings suggest an impaired 14 gating system, like that of the F400A mutant of SpFabF, that can no longer coordinate substrate transfer and C-C bond formation in the correct order. While gatekeeping elements appear to be a general feature of condensing enzymes based upon primary sequence analysis, a more precise stereochemical understanding of these elements will require further biochemical, structural, and computational interrogation.…”

Section: Additionally Ligand-bound and Acyl-enzyme Intermediate Ks Smentioning

confidence: 99%

Gating Mechanism of β-Ketoacyl-ACP Synthases

Mindrebo

Patel²,

Kim³

et al. 2019

Preprint

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Formation of carbon-carbon bonds via b-ketoacyl-acyl carrier protein (ACP) synthases (KS), are key reactions during de novo fatty acid and polyketide biosynthesis. KSs recognize multiple ACPs and choreograph ping-pong mechanisms often in an iterative fashion. Therefore, KSs must limit non-productive protein-protein interactions (PPIs) to achieve high degrees of reaction fidelity. To better understand the stereochemical features governing substrate discrimination during ACP•KS PPIs, we determined x-ray crystal structures complemented by molecular dynamic simulations of E. coli AcpP in complex with the elongating KSs, FabF and FabB. Covalently trapped substrate analogs were used to interrogate critical catalytic events accompanying carbon-carbon bond formation revealing a previously unknown gating mechanism during the binding and delivery of acyl-AcpPs. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features generally in elongating KSs. 3 Elaboration of these natural products is facilitated by a family of small ~8 kDa proteins, acyl carrier proteins (ACPs), that shuttle fatty acid and polyketide substrates and intermediates as they are processed by catalytic domains or discrete enzymes. 6,7 ACPs are translated in inactive apo forms and are posttranslationally modified to active holo forms with a 4'-phosphopantetheine (PPant) arm attached at a conserved serine residue, providing a thiol moiety that ligates substrates and intermediates to the ACPs. 6 Protein-protein interactions (PPIs) between ACPs and their enzymatic partners (or domains) regulate the catalytic activities of FASs and PKSs kinetically and stereochemically. 3,[8][9][10][11][12][13] Therefore, it is important to elucidate the structural and biophysical underpinnings of the PPIs between ACPs and their cognate KSs to understand reaction trajectories and product fidelity in FAS and PKS biosynthetic pathways.In contrast to initiating KSs 14,15 , which prime holo-ACPs, elongating KSs, can iteratively process fatty acid or polyketide intermediates. 3,16 Despite their distinct biosynthetic roles, these KSs operate via a common reaction mechanism, which can be separated into two halfreactions. 17,18 In the first half-reaction, an acyl-ACP associates with a KS, and its acyl cargo is transferred from the prosthetic PPant arm to a conserved, active site cysteine, producing a covalent acyl-KS intermediate and holo-ACP. Malonyl-ACP then replaces holo-ACP and undergoes a decarboxylative Claisen-like condensation with the acyl-KS adduct, producing and offloading β-ketoacyl-ACP 17 ( Figure S1). During the course of this ping-pong process, two distinct ACPs must sequentially interact with the KS enforcing exquisite temporal and spatial discrimination among subtly different chemical states of these acyl-ACPs.The Escherichia coli type II FAS system has served as a model for understanding ACPmediated PPIs as well as the enzymatic transformations catalyzed by FASs and PKSs ( Figure ...

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“…A report on bacterial fatty acid synthases (FabF) suggested that the H3364 counterpart is not an absolute necessity for the C−C bond‐forming reaction, but enhances the overall rate of the reaction . Analogous mutations of the catalytic His residues of the KS of 6‐deoxyerythronolide B synthase (DEBS) module 1 and E. coli FabB also resulted in moderate to very low turnover of the substrate . A double mutant (H3364A and H3404A) stalled the formation of 5 , thus proving that these residues are indispensable for the Michael addition.…”

Section: Figurementioning

confidence: 99%

“…A double mutant (H3364A and H3404A) stalled the formation of 5 , thus proving that these residues are indispensable for the Michael addition. According to functional studies with homologous KS domains, H3404 activates the malonyl carboxylate and stabilizes the enolate. Reduction of the rates in the carbon–carbon bond formation as well as lactonization (Figure C and A, v) indicates that H3404 plays a major role in all steps of the reaction.…”

Section: Figurementioning

confidence: 99%

On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

et al. 2018

View full text Add to dashboard Cite

Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In stark contrast, the KS of the rhizoxin PKS branching module mediates a Michael addition, which sets the basis for a pharmacophoric δ‐lactone moiety. The precise role of the KS was evaluated by site‐directed mutagenesis, chemical probes, and biotransformations. Biochemical and kinetic analyses helped to dissect branching and lactonization reactions and unequivocally assign the entire sequence to the KS. Probing the range of accepted substrates with diverse synthetic surrogates in vitro, we found that the KS tolerates defined acyl chain lengths to produce five‐ to seven‐membered lactones. These results show that the KS is multifunctional, as it catalyzes β‐branching and lactonization. Information on the increased product portfolio of the unusual, TE‐independent on‐line cyclization is relevant for synthetic biology approaches.

show abstract

Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase

Cited by 62 publications

References 19 publications

On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

Gating Mechanism of β-Ketoacyl-ACP Synthases

On‐Line Polyketide Cyclization into Diverse Medium‐Sized Lactones by a Specialized Ketosynthase Domain

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