EC Number   |
Protein Variants |
Reference |
|---|
  1.5.1.38 | K167A |
the mutant has apparently greatly increased Km and severely reduced kcat/Km values for NADPH and leads to a slight increase in kcat/Km for NADH |
724343 |
| 1.5.1.38 | more |
generation of a Y118 deletion mutant, DELTAY118, and of point mutants Y118S and Y118F. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintains the Pi-Pi stacking interactions at the tetramer interface and has kinetic parameters similar to those of wild-type SsuE. Substitution of the Pi-helical residue (Tyr118) to Ala or Ser transforms the enzymes into flavin-bound SsuE variants that can no longer support flavin reductase and desulfonation activities. These variants exist as dimers and can form protein-protein interactions with SsuD even though flavin transfer is not sustained. The DELTAY118 SsuE variant is flavin-free as purified and does not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of DELTAY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE Pi-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD. A 10fold lower binding affinity for flavin binding is observed with the DELTAY118 SsuE deletion variant than with wild-type SsuE. Although the DELTAY118 SsuE variant is unable to support NADPH oxidase activity, the 10fold decrease in flavin affinity would not account for the absence of activity because the flavin should still bind at the saturating concentrations of FMN used in the flavin reductase assays. Inability of Y118A, Y118S, and DELTAY118 SsuE to support desulfonation in the coupled assay |
764152 |
| 1.5.1.38 | N134A |
the mutant shows strongly decreased kcat/Km for NADPH |
724343 |
| 1.5.1.38 | R133A |
the mutant shows strongly decreased kcat/Km for NADPH |
724343 |
| 1.5.1.38 | R15A |
the mutant has apparently greatly increased Km and severely reduced kcat/Km values for NADPH |
724343 |
| 1.5.1.38 | R225A |
the mutant shows decreased kcat/Km for NADPH |
724343 |
| 1.5.1.38 | Y118A |
site-directed mutagenesis, altered kinetics compared to wild-type, inability of Y118A SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type |
764152 |
| 1.5.1.38 | Y118A |
site-directed mutagenesis, the SsuE variant converts the typically flavin-free enzyme to a flavin-bound form. The Y118A SsuE FMN cofactor is reduced with approximately 1 equiv of NADPH in anaerobic titration experiments, and the flavin remains bound following reduction. No measurable sulfite product is formed in a coupled assays with the Y118A SsuE variant and SsuD, demonstrating that flavin transfer is no longer supported |
741937 |
| 1.5.1.38 | Y118F |
site-directed mutagenesis, altered kinetics compared to wild-type.The mutant forms tetramers like the wild-type |
764152 |
| 1.5.1.38 | Y118S |
site-directed mutagenesis, inability of Y118S SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type |
764152 |
