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1.
Biochim Biophys Acta Gen Subj ; 1865(5): 129848, 2021 05.
Article in English | MEDLINE | ID: mdl-33460770

ABSTRACT

BACKGROUND: Environmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes. METHODS: In the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized. RESULTS: A phylogenetic analysis of the catalytic domain of pro- and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome. CONCLUSIONS: The characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity. GENERAL SIGNIFICANCE: The identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.


Subject(s)
Bacteria/genetics , Bacterial Proteins/genetics , Glycoside Hydrolases/genetics , Bacteria/metabolism , Bacterial Proteins/metabolism , Genome, Bacterial , Glycoside Hydrolases/metabolism , Hydrolysis , Lignin/metabolism , Metagenome , Phylogeny , Substrate Specificity
2.
Sci Rep ; 8(1): 17254, 2018 11 22.
Article in English | MEDLINE | ID: mdl-30467384

ABSTRACT

Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds. The recombinant purified protein produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro. A shorter protein version devoid of its N-terminal putative chloroplast transit peptide, Δ46AtDGAT3, was more stable in vitro, allowing biochemical and spectroscopic characterization. The results obtained demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Diacylglycerol O-Acyltransferase/chemistry , Diacylglycerol O-Acyltransferase/metabolism , Escherichia coli/growth & development , Arabidopsis/chemistry , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Chloroplasts/chemistry , Chloroplasts/metabolism , Diacylglycerol O-Acyltransferase/genetics , Escherichia coli/genetics , Germination , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Protein Domains , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Seeds/metabolism , Seeds/physiology , Thioredoxins/metabolism
3.
J Proteomics ; 169: 87-98, 2017 10 03.
Article in English | MEDLINE | ID: mdl-28918933

ABSTRACT

Lipid droplets are the major stock of lipids in oleaginous plant seeds. Despite their economic importance for oil production and biotechnological issues (biofuels, lubricants and plasticizers), numerous questions about their formation, structure and regulation are still unresolved. To determine water accessible domains of protein coating at lipid droplets surface, a structural proteomic approach has been performed. This technique relies on the millisecond timescale production of hydroxyl radicals by the radiolysis of water using Synchrotron X-ray white beam. Thanks to the evolution of mass spectrometry analysis techniques this approach allows the creation of a map of the solvent accessibility for proteins difficult to study by other means. Using these results, a S3 oleosin water accessibility map is proposed. This is the first time that such a map on an oleosin co-purified with plant lipid droplets and other associated protein is presented. BIOLOGICAL SIGNIFICANCE: Lipid droplet associated proteins function is linked to stability, structure and probably formation and lipid mobilization of droplets. Structure of these proteins in their native environment, at the interface between bulk water and the lipidic core of these organelles is only based on hydrophobicity plot. Using hydroxyl radical footprinting and proteomics approaches we studied water accessibility of one major protein in these droplets: S3 oleosin of Arabidopsis thaliana seeds.


Subject(s)
Arabidopsis Proteins/analysis , Lipid Droplets/chemistry , Plant Proteins/chemistry , Proteomics/methods , Hydroxyl Radical , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Water/metabolism
4.
PLoS One ; 10(11): e0143113, 2015.
Article in English | MEDLINE | ID: mdl-26581109

ABSTRACT

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.


Subject(s)
Arecaceae/enzymology , Diacylglycerol O-Acyltransferase/genetics , Diacylglycerol O-Acyltransferase/metabolism , Fatty Acids/metabolism , Gene Expression , Yarrowia/genetics , Amino Acid Motifs , Amino Acid Sequence , Arabidopsis/enzymology , Chromatography, Thin Layer , Conserved Sequence , Diacylglycerol O-Acyltransferase/chemistry , Lipid Droplets/metabolism , Lipid Metabolism , Microsomes/enzymology , Molecular Sequence Annotation , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/metabolism , Proteome/metabolism , Recombination, Genetic/genetics , Sequence Homology, Amino Acid , Transformation, Genetic , Triglycerides/metabolism
5.
PLoS One ; 9(3): e92237, 2014.
Article in English | MEDLINE | ID: mdl-24663078

ABSTRACT

Diacylglycerol acyltransferases (DGATs) catalyze the final and only committed step of triacylglycerol synthesis. DGAT activity is rate limiting for triacylglycerol accumulation in mammals, plants and microbes. DGATs belong to three different evolutionary classes. In Arabidopsis thaliana, DGAT1, encoded by At2g19450, is the major DGAT enzyme involved in triacylglycerol accumulation in seeds. Until recently, the function of DGAT2 (At3g51520) has remained elusive. Previous attempts to characterize its enzymatic function by heterologous expression in yeast were unsuccessful. In the present report we demonstrate that expression of a codon-optimized version of the DGAT2 gene is able to restore neutral lipid accumulation in the Saccharomyces cerevisiae mutant strain (H1246), which is defective in triacylglycerol biosynthesis. Heterologous expression of codon-optimized DGAT2 and DGAT1 induced the biogenesis of subcellular lipid droplets containing triacylglycerols and squalene. Both DGAT proteins were found to be associated with these lipid droplets. The fatty acid composition was affected by the nature of the acyltransferase expressed. DGAT2 preferentially incorporated C16:1 fatty acids whereas DGAT1 displayed preference for C16:0, strongly suggesting that these enzymes have contrasting substrate specificities.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Diacylglycerol O-Acyltransferase/genetics , Diacylglycerol O-Acyltransferase/metabolism , Saccharomyces cerevisiae/genetics , Codon/genetics , Gene Expression , Intracellular Space/metabolism , Lipid Droplets/metabolism , Mutation , Protein Transport , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Triglycerides/biosynthesis
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