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The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.

ABSTRACT: All members of the sulfotransferase (SOT, EC 2.8.2.-) protein family transfer a sulfuryl group from the donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxyl group of several classes of substrates. The primary structure of these enzymes is characterized by a histidine residue in the active site, defined PAPS binding sites and a longer SOT domain. Proteins with this SOT domain occur in all organisms from all three domains, usually as a multi-protein family. Arabidopsis thaliana SOTs, the best characterized SOT multi-protein family, contains 21 members. The substrates for several plant enzymes have already been identified, such as glucosinolates, brassinosteroids, jasmonates, flavonoids, and salicylic acid. Much information has been gathered on desulfo-glucosinolate (dsGl) SOTs in A. thaliana. The three cytosolic dsGl SOTs show slightly different expression patterns. The recombinant proteins reveal differences in their affinity to indolic and aliphatic dsGls. Also the respective recombinant dsGl SOTs from different A. thaliana ecotypes differ in their kinetic properties. However, determinants of substrate specificity and the exact reaction mechanism still need to be clarified. Probably, the three-dimensional structures of more plant proteins need to be solved to analyze the mode of action and the responsible amino acids for substrate binding. In addition to A. thaliana, more plant species from several families need to be investigated to fully elucidate the diversity of sulfated molecules and the way of biosynthesis catalyzed by SOT enzymes.

SUBMITTER: Hirschmann F

PROVIDER: S-EPMC4199319 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.

Hirschmann Felix F Krause Florian F Papenbrock Jutta J

Frontiers in plant science 20141016

All members of the sulfotransferase (SOT, EC 2.8.2.-) protein family transfer a sulfuryl group from the donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxyl group of several classes of substrates. The primary structure of these enzymes is characterized by a histidine residue in the active site, defined PAPS binding sites and a longer SOT domain. Proteins with this SOT domain occur in all organisms from all three domains, usually as a multi-protein family. Arabidopsis tha ...[more]

PMID: 25360143

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Project description:BackgroundACYL-LIPID THIOESTERASES (ALTs) are a subclass of plastid-localized, fatty acyl-acyl carrier protein (ACP) thioesterase enzymes from plants. They belong to the single hot dog-fold protein family. ALT enzymes generate medium-chain (C6-C14) and C16 fatty acids, methylketone precursors (β-keto fatty acids), and 3-hydroxy fatty acids when expressed heterologously in E. coli. The diverse substrate chain-length and oxidation state preferences of ALTs set them apart from other plant acyl-ACP thioesterases, and ALTs show promise as metabolic engineering tools to produce high-value medium-chain fatty acids and methylketones in bacterial or plant systems. Here, we used a targeted motif-swapping approach to explore connections between ALT protein sequence and substrate specificity. Guided by comparative motif searches and computational modelling, we exchanged regions of amino acid sequence between ALT-type thioesterases from Arabidopsis thaliana, Medicago truncatula, and Zea mays to create chimeric ALT proteins.ResultsComparing the activity profiles of chimeric ALTs in E. coli to their wild-type counterparts led to the identification of interacting regions within the thioesterase domain that shape substrate specificity and enzyme activity. Notably, the presence of a 31-CQH[G/C]RH-36 motif on the central α-helix was shown to shift chain-length specificity towards 12-14 carbon chains, and to be a core determinant of substrate specificity in ALT-type thioesterases with preference for 12-14 carbon 3-hydroxyacyl- and β-ketoacyl-ACP substrates. For an ALT containing this motif to be functional, an additional 108-KXXA-111 motif and compatible sequence spanning aa77-93 of the surrounding β-sheet must also be present, demonstrating that interactions between residues in these regions of the catalytic domain are critical to thioesterase activity. The behaviour of chimeric enzymes in E. coli also indicated that aa77-93 play a significant role in dictating whether an ALT will prefer ≤10-carbon or ≥ 12-carbon acyl chain-lengths, and aa91-96 influence selectivity for substrates of fully or partially reduced oxidation states. Additionally, aa64-67 on the hot dog-fold β-sheet were shown to be important for enabling an ALT to act on 3-hydroxy fatty acyl-ACP substrates.ConclusionsBy revealing connections between thioesterase sequence and substrate specificity, this study is an advancement towards engineering recombinant ALTs with product profiles suited for specific applications.

Dataset Information

The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.

Publications

The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.

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