Structural Basis for Binding Specificity between Subclasses of Modular Polyketide Synthase Docking Domains

Buchholz, Tonia J.; Geders, Todd W.; Bartley, F.E.; Reynolds, Kevin A.; Smith, Janet L.; Sherman, David H.

doi:10.1021/cb8002607

Cited by 113 publications

(204 citation statements)

References 49 publications

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“…A critical question is whether the ACPs engage HMGS simultaneously or sequentially. The affinity of HMGS for ACP D is tenfold greater than a native docking domain pair (K d = 0.5 μM vs. 5-25 μM) (20,21). HMGS has lower affinity for ACP A than for ACP D , based on the K d of 150-200 μM for the bryostatin HMGS and its cognate ACP A (15).…”

Section: Discussionmentioning

confidence: 98%

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Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

Maloney

Gerwick

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (K d = 0.5 μM) and could not be substituted by the acceptor ACP. Highresolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre-and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.natural products | polyketide synthase | curacin | HMG synthase | acyl carrier protein P olyketides are a large and chemically diverse group of natural products that includes many pharmaceuticals with a broad range of biological activities and applications as antibiotics, antifungals, antiinflammatory drugs, and cancer chemotherapeutic agents (1). Polyketide synthase (PKS) biosynthetic pathways are subjects of efforts to engineer diversification of natural products in pursuit of pharmaceutical leads and compounds of industrial importance (2). They are rich sources for development of chemoenzymatic catalysts based on PKS enzymes with unusual catalytic activities.Modular type I PKS pathways, among the most versatile of nature's systems, are biosynthetic assembly lines composed of modules that act in a defined sequence to produce complex products with a variety of functional groups and chiral centers. Each module is a set of fused catalytic domains that extend and modify a polyketide intermediate. Biosynthesis proceeds from intermediates tethered to acyl carrier protein (ACP) domains via a thioester link to a phosphopantetheine (Ppant) cofactor. A ketosynthase (KS) domain catalyzes extension of the intermediate, and subsequent modification domains typically catalyze reduction and/or methylation of the β-keto (3-keto) extension product. Beyond the enzymes for these core reactions, many PKS pathways also include other catalytic functionality. Among the most interesting of these noncanonical capabilities is alkylation at the β position by a set of β...

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Section: Discussionmentioning

confidence: 98%

“…The homologous KS extension enzymes distinguish their cognate donor and acceptor ACPs through two active site entrances (19) in interactions facilitated by fusion or noncovalent interaction of appended docking domains (20,21), which are absent in HMGS. (Table 1).…”

Section: Significancementioning

confidence: 99%

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

Maloney

Gerwick

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, the benchmark bimodular derivative of DEBS consists of LDD(4), (5)M1(2), and (3)M2ϩTE, where parenthetical numbers refer to the parent DEBS modules that are normally adjacent to the corresponding N-or C-terminal docking domains). These helical docking domains mediate weak but specific non-covalent interactions between donor and acceptor modules (K D ϭ ϳ100 M) (20).…”

Section: Engineering Of Chimeric Bimodular and Trimodular Pkss-mentioning

confidence: 99%

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Klaus

Ostrowski

Austerjost

et al. 2016

Journal of Biological Chemistry

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The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

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“…Three more-complex modules include up to three reductive domains (keto reductase, dehydratase, enoyl reductase) which sequentially modify the chemical form of the added monomer. The assembly of modules into the synthetic complex is directed by specific interactions between C-and N-terminal docking domains (6)(7)(8)(9)(10) as shown in Fig. 1.…”

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confidence: 99%

Emergent gene order in a model of modular polyketide synthases

Callahan

Thattai

Shraiman

2009

Proc. Natl. Acad. Sci. U.S.A.

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Polyketides are a class of biologically active heteropolymers produced by assembly line-like multiprotein complexes of modular polyketide synthases (PKS). The polyketide product is encoded in the order of the PKS proteins in the assembly line, suggesting that polyketide diversity derives from combinatorial rearrangement of these PKS complexes. Remarkably, the order of PKS genes on the chromosome follows the order of PKS proteins in the assembly line: This fact is commonly referred to as "collinearity". Here we propose an evolutionary origin for collinearity and demonstrate the mechanism by using a computational model of PKS evolution in a population. Assuming continuous evolutionary pressure for novel polyketides, and that new polyketide pathways are formed by horizontal transfer/recombination of PKS-encoding DNA, we demonstrate the existence of a broad range of parameters for which collinearity emerges spontaneously. Collinearity confers no fitness advantage in our model; it is established and maintained through a "secondary selection" mechanism, as a trait which increases the probability of forming long, novel PKS complexes through recombination. Consequently, collinearity hitchhikes on the successful genotypes which periodically sweep through the evolving population. In addition to computer simulation of a simplified model of PKS evolution, we provide a mathematical framework describing the secondary selection mechanism, which generalizes beyond the context of the present model. polyketides | horizontal transfer | evolution | collinearity | evolvability

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Structural Basis for Binding Specificity between Subclasses of Modular Polyketide Synthase Docking Domains

Cited by 113 publications

References 49 publications

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Emergent gene order in a model of modular polyketide synthases

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