EC Number   |
Protein Variants |
Reference |
|---|
   1.5.1.33 | K199E |
The mutant enzymes Y38D, Y195F, Y195W, and K199R are inactive even if they are purified as tetramers |
484954 |
| 1.5.1.33 | more |
a Leishmania infantum DHFR-TS null mutant is thymidine auxotroph showing a phenotype that can be rescued by the addition of thymidine or by transfection of the flavin-dependent bacterial thymidylate synthase (TS) gene ThyX. In DHFR-TS null mutants, it is impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 are provided episomally. Episomal expression of PTR1 in these cells does not revert thymidine auxotrophy. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that the results observed with the insect stage parasites are also replicated with intracellular parasites, it suggests that antifolate therapy in Leishmania only works if both DHFRTS and PTR1 are targeted simultaneously. Generation of a PTR1 null mutant in the DHFR-TSNEO/HYG cells using puromycin (PURO) and zeocin (ZEO) inactivation cassettes. Transfection of the PURO cassette in DHFR-TSNEO/HYG cloned cells leads to PTR1PURO/+ parasites. Transfection of the ZEO cassette in these parasites leads to the correct integration at the PTR1 locus but with a remaining intact copy of PTR1 in the population of cells analyzed. DHFR-TSNEO/HYG PTR1PURO/+ cells are complemented with episomally-expressed ThyX. These cells are able to grow in the absence of thymidine supplementation. Upon the transfection of the ZEO PTR1-inactivation cassette integration at the right locus is ontained but the hybridization signals are consistent with the population of parasites having the PTR1PURO/ZEO/+ genotype. This PTR1PURO/ZEO/+ genotype is also obtained in an independent PTR1 gene knockout experiment where ThyX is episomally expressed. When the genetic complementation is done with DHFR-TS instead of ThyX, the transfection of the ZEO PTR1-inactivation leads to a PTR1 chromosomal null mutant |
-, 765644 |
| 1.5.1.33 | more |
mutational analysis of the PruA YX3K motif. Generation of an Agrobacterium. tumefaciens DELTApruA strain |
-, 765008 |
| 1.5.1.33 | R24A |
site-directed mutagenesis, reduced activity compared to wild-type |
-, 765008 |
| 1.5.1.33 | R24G |
site-directed mutagenesis, highly reduced activity compared to wild-type |
765008 |
| 1.5.1.33 | Y161A |
site-directed mutagenesis, slightly reduced activity compared to wild-type |
-, 765008 |
| 1.5.1.33 | Y161A/Y163F |
site-directed mutagenesis, inactive mutant |
765008 |
| 1.5.1.33 | Y161F/Y163F |
site-directed mutagenesis, reduced activity compared to wild-type |
765008 |
| 1.5.1.33 | Y163A |
site-directed mutagenesis, inactive mutant |
-, 765008 |
| 1.5.1.33 | Y163F |
site-directed mutagenesis, slightly reduced catalytic efficiency but increased activity compared to wild-type |
-, 765008 |
