Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites

Perchat, Nadia; Saaidi, Pierre‐Loïc; Darii, Ekaterina; Pelle, Christine; Petit, Jean-Louis; Besnard-Gonnet, Marielle; Berardinis, Véronique de; Dupont, Maeva; Gimbernat, Alexandra; Salanoubat, Marcel; Fischer, Cécile; Perret, Alain

doi:10.1073/pnas.1722368115

Cited by 33 publications

(28 citation statements)

References 70 publications

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“…Finally, the abundance of carnitine in sea can be linked to the presence of the gene cluster in genera such as Oceanibulbus, Nisaea, and Leucothrix. These observations, which indicate that L-carnitine is mainly metabolized by Alphaproteobacteria and Betaproteobacteria, are evocative of what was observed for the degradation of another environmental abundant quaternary ammonium osmolyte, trigonelline (46).…”

Section: Discussionmentioning

confidence: 61%

Characterization ofl-Carnitine Metabolism in Sinorhizobium meliloti

et al. 2019

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L-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from ␥-butyrobetaine (GBB) and can be used as a carbon source. L-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of L-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of L-carnitine biosynthesis and degradation in Sinorhizobium meliloti. The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of L-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti. To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of L-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of L-carnitine as a ubiquitous nutrient.IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of L-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.

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Section: Discussionmentioning

confidence: 61%

Characterization ofl-Carnitine Metabolism in Sinorhizobium meliloti

et al. 2019

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“…As plant metabolites provide indispensable resources for human nutrition, energy and medicine (Butelli et al ., ; Chen et al ., ), dissecting the mechanism of metabolite biosynthesis in plants draws extreme interest (Saito and Matsuda, ; Cardoso et al ., ; Quadrana et al ., ; Zhao et al ., ; Fernie and Tohge, ; Perchat et al ., ; Tian et al ., ). In recent years, the rapid development of analysis approaches for metabolomes and multiomics techniques have greatly improved our knowledge of the naturally occurring metabolic variation in plants and its underlying genetic determinants in several species (Keurentjes et al ., ; Shang et al ., ; Sadre et al ., ; Tohge et al ., ; Wen et al ., ; Fernie and Tohge, ; Rai et al ., ; Westhues et al ., ; Xiao et al ., ; Zhu et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population

Wang

Gong

et al. 2019

The Plant Journal

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Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and L-asparagine content variation across populations, respectively. MetaboliteÀagronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metaboliteÀagronomic trait relationship and the corresponding genetic basis.

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“…Purification of IGCAmDH1, IGCAmDH5, Sgor AmDH, Chat AmDH, Tther AmDH, Acol AmDH, MATOUAmDH1, and MATOUAmDH2 were conducted from a 500 mL culture by nickel affinity chromatography in tandem with gel filtration (Hi Load 16/600 Superdex 200 pg) as described previously . The storage buffer was 50 mM Tris.HCl pH 8.0, 50 mM NaCl, 15% glycerol and 1 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

Metagenomic Mining for Amine Dehydrogenase Discovery

et al. 2020

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Amine dehydrogenases (AmDHs) catalyze the enzymatic reduction of ketones to amines, serving as a suitable biocatalytic route for amine synthesis. A limited number of experimentally validated native AmDHs (nat‐AmDHs) have been reported recently, expanding the sequences with this function to complement the small set of engineered enzymes. Since researchers can now probe into the vast diversity of enzymes within niche environments by a metagenomics approach, a tandem metagenomic and bioinformatic approach is a powerful tool to identify new members of limited enzyme families to access new features in an iterative fashion. The previously untapped biocatalytic reservoirs of the ocean environment and human microbiome were screened for potential AmDHs using a hidden Markov model. Among the hundreds of hits, a subset of 18 enzymes was selected for further characterization and were confirmed to display AmDH activity. Additional analysis on six enzymes confirmed altered cofactor specificities and variation in substrate scopes, catalytic efficiencies, and active site residues compared to the reference nat‐AmDHs previously described. Particularly, MATOUAmDH2 from an eukaryotic organism demonstrated specific activity of 11.07 and 0.88 U mg−1 toward isobutyraldehyde and 1,2‐cyclohexadione respectively. Their abundance among the screened environments was also described. The protein sequence diversity of validated AmDHs reached by this metagenomics mining strategy highlights the success of such an approach. Metagenomically mined proteins, including eukaryotic ones, stand to increase the reach of biocatalysis towards enviromentally benign processes.

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Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites

Cited by 33 publications

References 70 publications

Characterization ofl-Carnitine Metabolism in Sinorhizobium meliloti

Characterization ofl-Carnitine Metabolism in Sinorhizobium meliloti

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population

Metagenomic Mining for Amine Dehydrogenase Discovery

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