Dissection of the neocarazostatin

a C4 alkyl side chain biosynthesis by in vitro reconstitution

Li Su, Rui Zhang, Kwaku Kyeremeh, Zixin Deng, Hai Deng (Corresponding Author), Yi Yu

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C4 alkyl side chain attached to ring "A". Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C4 unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of β-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C4 alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.

Original languageEnglish
Pages (from-to)3843-3848
Number of pages6
JournalOrganic & Biomolecular Chemistry
Volume15
Issue number18
Early online date7 Apr 2017
DOIs
Publication statusPublished - 14 May 2017

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Dissection
dissection
biosynthesis
Biosynthesis
Acyl Carrier Protein
enzymes
holo-(acyl-carrier-protein) synthase
fatty acids
proteins
Enzymes
Fatty Acids
Free Radical Scavengers
Acetyl Coenzyme A
carbazoles
Biosynthetic Pathways
metabolism
Multigene Family
NADP
Metabolism
genes

Cite this

Dissection of the neocarazostatin : a C4 alkyl side chain biosynthesis by in vitro reconstitution. / Su, Li; Zhang, Rui; Kyeremeh, Kwaku; Deng, Zixin; Deng, Hai (Corresponding Author); Yu, Yi.

In: Organic & Biomolecular Chemistry, Vol. 15, No. 18, 14.05.2017, p. 3843-3848.

Research output: Contribution to journalArticle

Su, Li ; Zhang, Rui ; Kyeremeh, Kwaku ; Deng, Zixin ; Deng, Hai ; Yu, Yi. / Dissection of the neocarazostatin : a C4 alkyl side chain biosynthesis by in vitro reconstitution. In: Organic & Biomolecular Chemistry. 2017 ; Vol. 15, No. 18. pp. 3843-3848.
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abstract = "Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C4 alkyl side chain attached to ring {"}A{"}. Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C4 unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of β-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C4 alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.",
author = "Li Su and Rui Zhang and Kwaku Kyeremeh and Zixin Deng and Hai Deng and Yi Yu",
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AU - Su, Li

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AU - Deng, Zixin

AU - Deng, Hai

AU - Yu, Yi

N1 - This work was supported by grants from the National Natural Science Foundation of China (31570033 to Y. Y.), the Fundamental Research Funds for the Central Universities (2042017kf0191 to Y. Y.), and the Leverhulme Trust-Royal Society Africa Award (AA090088 to H. D.)

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N2 - Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C4 alkyl side chain attached to ring "A". Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C4 unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of β-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C4 alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.

AB - Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C4 alkyl side chain attached to ring "A". Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C4 unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of β-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C4 alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.

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DO - 10.1039/c7ob00617a

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JO - Organic & Biomolecular Chemistry

JF - Organic & Biomolecular Chemistry

SN - 1477-0520

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