A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Haines, Anthony S.; Xu, Dong; Song, Zhiyong; Farmer, Rohit; Williams, Christopher; Hothersall, Joanne; Płoskoń, Eliza; Wattana-Amorn, Pakorn; Stephens, Elton R.; Yamada, Erika; Gurney, Rachel; Takebayashi, Yuiko; Masschelein, Joleen; Cox, Russell J.; Lavigne, Rob; Willis, Christine L.; Simpson, Thomas J.; Crosby, John; Winn, Peter J.; Thomas, Christopher M.; Crump, Matthew P.

doi:10.1038/nchembio.1342

Cited by 59 publications

(93 citation statements)

References 59 publications

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“…Intriguingly, short helical turns similar to H II ’ have been proposed to contribute to other ACP interactions. 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).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the ACP specificity of trans-AT’s needs to be better understood so that specificity can be manipulated. Although evidence of the ability to manipulate ACP interactions with other domains is emerging (Haines, et al, 2013; Kapur, et al, 2011; Kapur, et al, 2012), little is known regarding trans-AT:ACP interactions. Indeed, including the malonyl-CoA-ACP transacylase (MCAT) that primarily serves ACP’s from type II fatty acid synthases, only two trans-AT structures are available (Keatinge-Clay, et al, 2003; Wong, et al, 2011), in addition to that of a bi-functional trans-AT/decarboxylase (Lohman, et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

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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“…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%

“…HMGS enzymes are highly selective for acetoacetyl-ACP A and acetyl-ACP D , and do not react with CoA substrates (15)(16)(17). At the sequence level, the donor and acceptor ACPs clade separately from each other and from nonbranch ACPs from the same pathways (15,18). Thus, by selecting for the correct acyl-ACPs, HMGS prevents the formation of aberrantly branched metabolites.…”

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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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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“…57). Nonetheless, the structures of all three of the core domains have now been reported: the KS domain (one from the rhizoxin pathway solved as a didomain with a downstream branching (B) domain 58 and another monodomain KS fragment from the bacillaene PKS 59 ), a discrete AT domain 57 and multiple ACP domains 60,61 . In addition, structures of domains for several unusual tailoring activities from the bacillaene assembly line (a KR that reduces both α-and β-keto groups 62 and an enoyl-isomerase (EI) that catalyzes a double-bond shift 63 ) have been reported.…”

Section: The Dissection Approach To Studying Functional Domainsmentioning

confidence: 97%

The structural biology of biosynthetic megaenzymes

Weissman¹

2015

Nat Chem Biol

177

165

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The modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are among the largest and most complicated enzymes in nature. In these biosynthetic systems, independently folding protein domains, which are organized into units called 'modules', operate in assembly-line fashion to construct polymeric chains and tailor their functionalities. Products of PKSs and NRPSs include a number of blockbuster medicines, and this has motivated researchers to understand how they operate so that they can be modified by genetic engineering. Beginning in the 1990s, structural biology has provided a number of key insights. The emerging picture is one of remarkable dynamics and conformational programming in which the chemical states of individual catalytic domains are communicated to the others, configuring the modules for the next stage in the biosynthesis. This unexpected level of complexity most likely accounts for the low success rate of empirical genetic engineering experiments and suggests ways forward for productive megaenzyme synthetic biology.

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A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Cited by 59 publications

References 59 publications

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

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

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

The structural biology of biosynthetic megaenzymes

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