Cloned (Comment) | Organism |
---|---|
gene ypdA, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 | Staphylococcus aureus |
Protein Variants | Comment | Organism |
---|---|---|
G10A | site-directed mutagenesis, the mutation completely abolishes cofactor binding activity as glycine 10 is the first glycine residue in a putative Rossman fold domain (GxGxxG), which is characteristic of cofactor binding enzymes and presumed to function in the reduction of oxidized bacillithiol disulfide (BSSB). The YpdA G10A protein is unable to consume NADPH or NADH. Carbohydrate metabolism is mostly down regulated in the mutant, reduced fitness of the ypdA mutant in the competitive fitness assays with the wild-type in chemical defined medium | Staphylococcus aureus |
additional information | mutation of gene ypdA by replacing the ORF with a kanamycin cassette through homologous recombination in an SH1000 strain corrected for BSH production, and construction of the complement strain by replacing the kanamycin cassette with the native ypdA gene, phenotypes, overview. No obvious growth defect of the mutant vs. the parent or the complemented mutant in TSB is detected. Fitness defects might be masked by other metabolic pathways in rich media that neutralize the effect of stress. Accordingly, the fitness of the ypdA mutant is evaluated by a competition assay which has been shown to be useful for teasing out subtle fitness defects between closely related strains. Despite identical growth kinetics of the mutant vs. the parent in complex media such as TSB and CDM, overnight competition assays reveal that the wild-type strain out-competes the ypdA mutant in competitive growth in CDM, while the competitive index does not change in TSB media. These results suggest that while the ypdA mutation has a limited effect on growth in complex media in vitro, the mutant exhibits a fitness defect vs. the wild-type when grown competitively in chemically defined medium. Cells overexpressing YpdA are able to survive better than cells with just the empty vector, and this difference in survival is abolished when oxidative burst of PMNs is blocked by DPI | Staphylococcus aureus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
oxidized bacillithiol disulfide + NADH + H+ | Staphylococcus aureus | - |
reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADH + H+ | Staphylococcus aureus NCTC 8325 | - |
reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADH + H+ | Staphylococcus aureus PS 47 | - |
reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | Staphylococcus aureus | - |
reduced bacillithiol disulfide + NADP+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | Staphylococcus aureus NCTC 8325 | - |
reduced bacillithiol disulfide + NADP+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | Staphylococcus aureus PS 47 | - |
reduced bacillithiol disulfide + NADP+ | - |
r | |
thioredoxin + NADP+ | Staphylococcus aureus | - |
thioredoxin disulfide + NADPH + H+ | - |
r | |
thioredoxin + NADP+ | Staphylococcus aureus NCTC 8325 | - |
thioredoxin disulfide + NADPH + H+ | - |
r | |
thioredoxin + NADP+ | Staphylococcus aureus PS 47 | - |
thioredoxin disulfide + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Staphylococcus aureus | Q2FYF3 | - |
- |
Staphylococcus aureus NCTC 8325 | Q2FYF3 | - |
- |
Staphylococcus aureus PS 47 | Q2FYF3 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21 | Staphylococcus aureus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
oxidized bacillithiol disulfide + NADH + H+ | - |
Staphylococcus aureus | reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADH + H+ | - |
Staphylococcus aureus NCTC 8325 | reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADH + H+ | - |
Staphylococcus aureus PS 47 | reduced bacillithiol disulfide + NAD+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | - |
Staphylococcus aureus | reduced bacillithiol disulfide + NADP+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | - |
Staphylococcus aureus NCTC 8325 | reduced bacillithiol disulfide + NADP+ | - |
r | |
oxidized bacillithiol disulfide + NADPH + H+ | - |
Staphylococcus aureus PS 47 | reduced bacillithiol disulfide + NADP+ | - |
r | |
thioredoxin + NADP+ | - |
Staphylococcus aureus | thioredoxin disulfide + NADPH + H+ | - |
r | |
thioredoxin + NADP+ | - |
Staphylococcus aureus NCTC 8325 | thioredoxin disulfide + NADPH + H+ | - |
r | |
thioredoxin + NADP+ | - |
Staphylococcus aureus PS 47 | thioredoxin disulfide + NADPH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
bacillithiol disulfide reductase | - |
Staphylococcus aureus |
YpdA | - |
Staphylococcus aureus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | required, bound | Staphylococcus aureus | |
additional information | purified YpdA can consume both NADPH and NADH. Cofactor binding activity by glycine 10, the first glycine residue in a putative Rossman fold domain (GxGxxG) | Staphylococcus aureus | |
NAD+ | - |
Staphylococcus aureus | |
NADH | - |
Staphylococcus aureus | |
NADP+ | - |
Staphylococcus aureus | |
NADPH | - |
Staphylococcus aureus |
Organism | Comment | Expression |
---|---|---|
Staphylococcus aureus | wild-type cells exposed to strong oxidants such as H2O2 and hypochlorous acid (HOCl), nitric oxide-generating NOC-12, the toxic electrophilic metabolic byproduct methylglyoxal (MG), and thiol stress-inducing diamide at experimentally determined non-lethal concentrations all show significant increases in fluorescence attributable to enhanced ypdA promoter activity, these increases in transcriptional activity result in enhanced YpdA protein expression, YpdA levels are induced by stress | up |
General Information | Comment | Organism |
---|---|---|
evolution | based on sequence analysis, YpdA belongs to the pyridine nucleotide-disulfide oxidoreductase family of proteins that use flavin adenine dinucleotide (FAD) to transport reducing equivalents from NAD(P)H to cysteine residues | Staphylococcus aureus |
malfunction | YpdA overexpression confers greater resistance to stress. The YpdA G10A mutant protein is unable to consume NADPH or NADH. Carbohydrate metabolism is mostly down regulated in the mutant, reduced fitness of the ypdA mutant in the competitive fitness assays with the wild-type in chemical defined medium. Decreased fitness of the ypdA mutant results in reduced survival in neutrophils (PMNs). The ypdA mutant survives less well than the parent and the complemented mutant. The survival of SH1000 (BSH null strain), akin to the ypdA mutant, is also less than the parent and complemented mutant in this assay | Staphylococcus aureus |
physiological function | enzyme YpdA participates in stress response upon exposure to specific physiologically relevant stresses. Role of YpdA in regulating BSH and BSSB levels, the enzyme is responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH). Enzyme YpdA plays in protecting Staphylococcus aureus cells from the oxidative killing by human neutrophils (PMNs) | Staphylococcus aureus |