4-Coumarate:coenzyme A ligase isoform 3 from <i>Piper nigrum</i> (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids
Bibliographic record
Abstract
Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| 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) | 0.001 | 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".