Dataset Information

Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis.

ABSTRACT: Despite the diversity of reactions catalyzed by 2-oxoglutarate-dependent nonheme iron (Fe/2OG) enzymes identified in recent years, only a limited number of these enzymes have been investigated in mechanistic detail. In particular, several Fe/2OG-dependent enzymes capable of catalyzing isocyanide formation have been reported. While the glycine moiety has been identified as a biosynthon for the isocyanide group, how the actual conversion is effected remains obscure. To elucidate the catalytic mechanism, we characterized two previously unidentified (AecA and AmcA) along with two known (ScoE and SfaA) Fe/2OG-dependent enzymes that catalyze N≡C triple bond installation using synthesized substrate analogues and potential intermediates. Our results indicate that isocyanide formation likely entails a two-step sequence involving an imine intermediate that undergoes decarboxylation-assisted desaturation to yield the isocyanide product. Results obtained from the in vitro experiments are further supported by mutagenesis, the product-bound enzyme structure, and in silico analysis.

SUBMITTER: Chen TY

PROVIDER: S-EPMC9390461 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Publications

Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis.

Chen Tzu-Yu TY Zheng Ziyang Z Zhang Xuan X Chen Jinfeng J Cha Lide L Tang Yijie Y Guo Yisong Y Zhou Jiahai J Wang Binju B Liu Hung-Wen HW Chang Wei-Chen WC

ACS catalysis 20220131 4

Despite the diversity of reactions catalyzed by 2-oxoglutarate-dependent nonheme iron (Fe/2OG) enzymes identified in recent years, only a limited number of these enzymes have been investigated in mechanistic detail. In particular, several Fe/2OG-dependent enzymes capable of catalyzing isocyanide formation have been reported. While the glycine moiety has been identified as a biosynthon for the isocyanide group, how the actual conversion is effected remains obscure. To elucidate the catalytic mech ...[more]

PMID: 35992736

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Project description:In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.

Dataset Information

Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis.

Publications

Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis.

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