Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol
Bibliographic record
Abstract
Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1's glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".