Application | Comment | Organism |
---|---|---|
industry | modification of coenzyme specificity and alteration of product enantioselectivity in SDRs by using the protein engineering approach, which will have valuable industrial applications | Candida parapsilosis |
Cloned (Comment) | Organism |
---|---|
wild-type and mutants expressed in Escherichia coli strain BL21(DE3) as His6-tagged proteins | Candida parapsilosis |
Protein Variants | Comment | Organism |
---|---|---|
H68D | produces (R)-enantiomer with low optical purity and yield | Candida parapsilosis |
H68D/P69D | produces (R)-enantiomer with low optical purity and yield | Candida parapsilosis |
P69D | produces (R)-enantiomer with low optical purity and yield | Candida parapsilosis |
S67D | produces (R)-enantiomer with low optical purity and yield | Candida parapsilosis |
S67D/H68D | produces (R)-1-phenyl-1,2-ethanediol with high optical purity of 95.4% and a yield of 83.1% in the NADH-linked reaction. It results in a nearly 10fold increase and a 20fold decrease in the kcat/Km value when NADH and NADPH are used as the cofactors, respectively, but maintaining a kcat value essentially the same with respect to wild-type. The mutant has a stronger preference for NADH and weaker binding for NADPH. It exhibits a secondary structure and melting temperature similar to the wild-type form. NADH provides maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converts the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability | Candida parapsilosis |
S67D/P69D | produces (R)-enantiomer with low optical purity and yield | Candida parapsilosis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.59 | - |
2-hydroxyacetophenone | wild-type, NADPH as cofactor | Candida parapsilosis | |
0.76 | - |
2-hydroxyacetophenone | mutant S67D/H68D, NADH as cofactor | Candida parapsilosis | |
1.22 | - |
2-hydroxyacetophenone | mutant P69D, NADH as cofactor | Candida parapsilosis | |
1.26 | - |
2-hydroxyacetophenone | mutant S67D, NADH as cofactor | Candida parapsilosis | |
1.35 | - |
2-hydroxyacetophenone | mutant H68D, NADH as cofactor | Candida parapsilosis | |
1.42 | - |
2-hydroxyacetophenone | mutant S67D/P69D, NADH as cofactor | Candida parapsilosis | |
1.46 | - |
2-hydroxyacetophenone | mutant H68D/P69D, NADH as cofactor | Candida parapsilosis | |
1.51 | - |
2-hydroxyacetophenone | mutant P69D, NADPH as cofactor | Candida parapsilosis | |
1.54 | - |
2-hydroxyacetophenone | mutant S67D, NADPH as cofactor | Candida parapsilosis | |
1.55 | - |
2-hydroxyacetophenone | mutant S67D/P69D, NADPH as cofactor | Candida parapsilosis | |
1.58 | - |
2-hydroxyacetophenone | mutant H68D, NADPH as cofactor | Candida parapsilosis | |
1.61 | - |
2-hydroxyacetophenone | mutant H68D/P69D, NADPH as cofactor | Candida parapsilosis | |
7.89 | - |
2-hydroxyacetophenone | wild-type, NADH as cofactor | Candida parapsilosis | |
10.27 | - |
2-hydroxyacetophenone | mutant S67D/H68D, NADPH as cofactor | Candida parapsilosis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
110000 | - |
analytical ultracentrifugation analysis, mutant S67D/H68D and wild-type | Candida parapsilosis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Candida parapsilosis | B2KJ46 | - |
- |
Purification (Comment) | Organism |
---|---|
by affinity chromatography on a Ni2+ column and by gel filtration | Candida parapsilosis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxyacetophenone + NADPH + H+ | - |
Candida parapsilosis | (S)-1-phenyl-1,2-ethanediol + NADP+ | product of wild-type enzyme, 96.2% purity | ? | |
2-hydroxyacetophenone + NADPH + H+ | - |
Candida parapsilosis | (R)-1-phenyl-1,2-ethanediol + NADP+ | product of mutant enzyme S67D/H68D, 95.4% purity | ? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | gel filtration, mutant S67D/H68D and wild-type | Candida parapsilosis |
Synonyms | Comment | Organism |
---|---|---|
SCR | - |
Candida parapsilosis |
short-chain carbonyl reductase | - |
Candida parapsilosis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.21 | - |
2-hydroxyacetophenone | mutant S67D/H68D, NADPH as cofactor | Candida parapsilosis | |
1.26 | - |
2-hydroxyacetophenone | mutant S67D/H68D, NADH as cofactor | Candida parapsilosis | |
1.32 | - |
2-hydroxyacetophenone | wild-type, NADH as cofactor | Candida parapsilosis | |
1.48 | - |
2-hydroxyacetophenone | wild-type, NADPH as cofactor | Candida parapsilosis | |
1.72 | - |
2-hydroxyacetophenone | mutant H68D/P69D, NADPH as cofactor | Candida parapsilosis | |
1.95 | - |
2-hydroxyacetophenone | mutant H68D, NADPH as cofactor | Candida parapsilosis | |
2.23 | - |
2-hydroxyacetophenone | mutant P69D, NADPH as cofactor | Candida parapsilosis | |
2.32 | - |
2-hydroxyacetophenone | mutant S67D/P69D, NADPH as cofactor | Candida parapsilosis | |
2.35 | - |
2-hydroxyacetophenone | mutant S67D, NADPH as cofactor | Candida parapsilosis | |
6.37 | - |
2-hydroxyacetophenone | mutant H68D/P69D, NADH as cofactor | Candida parapsilosis | |
6.54 | - |
2-hydroxyacetophenone | mutant P69D, NADH as cofactor | Candida parapsilosis | |
6.58 | - |
2-hydroxyacetophenone | mutant H68D, NADH as cofactor | Candida parapsilosis | |
6.63 | - |
2-hydroxyacetophenone | mutant S67D, NADH as cofactor | Candida parapsilosis | |
6.76 | - |
2-hydroxyacetophenone | mutant S67D/P69D, NADH as cofactor | Candida parapsilosis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD(P)H | exhibits coenzyme specificity for NADPH over NADH | Candida parapsilosis |