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Literature summary for 1.1.1.184 extracted from

  • Zhang, R.; Xu, Y.; Sun, Y.; Zhang, W.; Xiao, R.
    Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction (2009), Appl. Environ. Microbiol., 75, 2176-2183.
    View publication on PubMedView publication on EuropePMC

Application

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(Commentary)

Cloned (Comment) Organism
wild-type and mutants expressed in Escherichia coli strain BL21(DE3) as His6-tagged proteins Candida parapsilosis

Protein Variants

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
110000
-
analytical ultracentrifugation analysis, mutant S67D/H68D and wild-type Candida parapsilosis

Organism

Organism UniProt Comment Textmining
Candida parapsilosis B2KJ46
-
-

Purification (Commentary)

Purification (Comment) Organism
by affinity chromatography on a Ni2+ column and by gel filtration Candida parapsilosis

Substrates and Products (Substrate)

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

Subunits Comment Organism
homotetramer gel filtration, mutant S67D/H68D and wild-type Candida parapsilosis

Synonyms

Synonyms Comment Organism
SCR
-
Candida parapsilosis
short-chain carbonyl reductase
-
Candida parapsilosis

Turnover Number [1/s]

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

Cofactor Comment Organism Structure
NAD(P)H exhibits coenzyme specificity for NADPH over NADH Candida parapsilosis