The Determinants of Activity and Specificity in Actinorhodin Type II Polyketide Ketoreductase

Javidpour, P.; Bruegger, Joel; Srithahan, Supawadee; Korman, Tyler P.; Crump, Matthew P.; Crosby, John; Burkart, Michael D.; Tsai, Shiou‐Chuan

doi:10.1016/j.chembiol.2013.07.016

Cited by 37 publications

(106 citation statements)

References 38 publications

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“…The Michaelis–Menten kinetic parameters were determined for 1 : K m =0.29±0.08 m m , k cat =59.2±5.6 min −1 (Figure S9). The kinetic parameters are comparable to those reported for the KRs from type I (LovB in lovastatin biosynthesis) and type II (ActKR in actinorhodin biosynthesis) PKS systems (Table S1). Iga13 also accepted NADH as a cofactor (Figure S8).…”

Section: Methodssupporting

confidence: 81%

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Katsuyama

Shin‐ya

et al. 2018

Angew Chem Int Ed

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While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β-keto group and dehydration of a β-hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11-Iga12), Iga13 and Iga16 with malonyl and hexanoyl-CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C , C , C , and C ), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto-reduction and dehydration with strict stereo-specificity. We propose "highly reducing type II PKS subfamily" for the polyene-producing type II PKSs.

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Section: Methodssupporting

confidence: 81%

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Katsuyama

Shin‐ya

et al. 2018

Angew Chem Int Ed

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“…However,I ga13 lacks any motifs that have been reported to determine the stereospecificity of KRs (LDD motif in type I PKSs [13] and PGG motif in type II PKSs). Thek inetic parameters are comparable to those reported for the KRs from type I (LovB in lovastatin biosynthesis) [15] and type II (ActKR in actinorhodin biosynthesis) [16] PKS systems (Table S1). Incubation of 1 with NADPH and Iga13 led to the formation of (3R)-hydroxyoctanoyl-NAC (2R; Figure 1b and Figure S8).…”

supporting

confidence: 80%

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Katsuyama

Shin‐ya

et al. 2018

Angewandte Chemie

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While type II polyketide synthases (PKSs) are knownfor producing aromatic compounds,aphylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures.H ere we report in vitro analysis of such atype II PKS,IgaPKS for ishigamide biosynthesis.The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a b-keto group and dehydration of a b-hydroxy group,r espectively,t of orm atrans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/ chain length factor complex (Iga11-Iga12), Iga13 and Iga16 with malonyl and hexanoyl-CoAs and NADPH followed by KOHhydrolysis resulted in the formation of four unsaturated carboxylic acids (C 8 ,C 10 ,C 12 ,and C 14 ), indicating that IgaPKS catalyzest etraene formation by repeating the cycle of condensation, keto-reduction and dehydration with strict stereospecificity.W epropose "highly reducing type II PKS subfamily" for the polyene-producing type II PKSs.

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“…Extensive biochemical analysis and docking studies suggest that KR interactions with the minimal PKS are essential during biosynthesis (33,34), which could explain why most clusters harbor either a C9 or C19 KR (although there are exceptions, such as resistomycin, the spore pigments, and anciently diverged orphans that harbor no KR). The C19-to C9-KR gene swap events can be seen at the bottom of Fig.…”

Section: Resultsmentioning

confidence: 99%

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

Hillenmeyer

Vandova

Berlew

et al. 2015

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

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Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.evolution | polyketide | natural products | gene cluster

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The Determinants of Activity and Specificity in Actinorhodin Type II Polyketide Ketoreductase

Cited by 37 publications

References 38 publications

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

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