Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Busche, Alena; Gottstein, Daniel; Hein, Christopher; Ripin, Nina; Pader, Irina; Tufar, Peter; Eisman, Eli B.; Gu, Liangcai; Walsh, Christopher T.; Sherman, David H.; Löhr, Frank; Güntert, Peter; Dötsch, Volker

doi:10.1021/cb200352q

Cited by 35 publications

(57 citation statements)

References 54 publications

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“…ACPs from PKS pathways (18,24,25,31) have distinctive surface charge distributions compared with FAS ACPs (26)(27)(28)(29), but in both systems the ACP helix II-III surface interacts with enzymes (Fig. S6).…”

Section: Discussionmentioning

confidence: 99%

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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: 99%

“…S6). We compared the surface features of other ACPs in complexes with cognate enzymes (24)(25)(26)(27)(28)(29) to ACP D (Fig. S6).…”

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, a short helix located within L II has been proposed to flag ACP’s for β-branching in polyketide biosynthesis (Haines, et al, 2013). In another example, a similarly short helical turn was implicated in the specific interaction between the curacin A ACP I and halogenase embedded within the CurA module (Busche, et al, 2012). Further, in the non-reducing PKS PksA ACP, an aspartate equivalent to ACP5 Kir D60, located in a small helical turn, is predicted to make a key electrostatic interaction with the KS domain of PksA (Bruegger, et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase

Musiol

Weber

et al. 2014

Chemistry & Biology

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SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues.

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“…The only structural information available regarding the interaction between enzyme and a CP-tethered substrate is an NMR structure of a CP domain from the curacin biosynthetic pathway and mapping of the CP residues that modulate Cur Hal activity based on mutational analyses. 276 However, the discovery of WelO5 has facilitated the attainment of a co-crystal structure with the substrate 12- epi -fischerindole U, 271 and furthered the elucidation of the mechanism of halogenation/hydroxylation control, as discussed below.…”

Section: Non-heme Iron-dependent Halogenasesmentioning

confidence: 99%

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

et al. 2017

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Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature’s halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

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Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Cited by 35 publications

References 54 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

Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

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