Characterization of the active site of Methionine γ-Lyase from Trichomonas vaginalis
Bibliographic record
Abstract
Resistance to, metronidazole and tinidazole, the drugs currently prescribed for the treatment of trichomoniasis, an infection of the genito-urinary tract of humans by the protozoan Trichomonas vaginalis, has created a need for the development of therapeutics with a different mode of action.Trifluoromethionine (TFM) has been proposed as a potential novel antiinfective prodrug that is activated by the enzyme methionine γ-lyase (MGL), which is present in T. vaginalis but not in the cells of the human host.The presence of closely related enzymes, such as cystathionine γ-lyase, in humans necessitates the development of TFM derivatives that are highly selective for TvMGL so that activation of the prodrug will be limited to the parasitic cells.An essential step in the development of selective TFM-based prodrugs is mapping of the TvMGL active site to identify the residues that participate in substrate binding and catalysis, particularly compared to related human enzymes.Therefore, the primary goal of this thesis was to determine the effect of a set of 9 site-directed variants of key active-site residues on the steady state kinetic parameters of TvMGL.As a prerequisite to this study, an affinity purification protocol and continuous assays for the methionine, homocysteine and cysteine hydrolysis activities of TvMGL were developed.The Y56F, R58A, Y111F, S338A, R373A and R373K variants of TvMGL lack detectable methionine and homocysteine hydrolysis activity and the turnover rate of the I55A and D239A variants is reduced up to 14-fold, while that of L339A is within 2-fold of the wild-type TvMGL enzyme.Roles for residue R373 in binding the α-carboxylate group of the substrate and for I55 in hydrophobic packing with iii the nonpolar side chain of the substrate are proposed.Residues Y56 and R58 are expected to anchor and position the PLP cofactor, through interaction with the phosphate moiety.Residue S338 may guide the catalytic lysine and Y111 is proposed to play a role in proton transfer, guided by interaction with R58, in the context of the α,γ-elimination reaction catalyzed by TvMGL.These results further our understanding of the substrate-binding surface of the TvMGL active site, information that will assist in the development of selective TFMbased prodrugs for the treatment of trichomoniasis. AATAspartate aminotransferase
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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) | 0.001 | 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".