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Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

ABSTRACT: Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates-S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates-was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes.

SUBMITTER: Kong W

PROVIDER: S-EPMC5003058 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

Kong Wenwen W Li Jing J Yu Qingyue Q Cang Wei W Xu Rui R Wang Yang Y Ji Wei W

Frontiers in plant science 20160829

Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates-S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates-was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, w ...[more]

PMID: 27621741

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Project description:Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and "fish" odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries.IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of "fish" caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.

Dataset Information

Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

Publications

Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

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