Cloned (Comment) | Organism |
---|---|
gene phaB, recombinant expression in Corynebacterium glutamicum strain ATCC 13803, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Cupriavidus necator |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His-tagged wild-type and mutant enzymes in complex with NADP+ and acetoacetyl-CoA, wild-type protein crystals grow from 0.1 M MES, pH 7.1, 0.9 mM NADP+, 0.9 mM acetoacetyl-CoA, 1.6 M ammonium sulfate, and 10% 1,4-dioxane, mutant crystals grow from 0.6-1.2 M sodium-potassium tartrate, 0.16-0.20 M lithium sulfate, and 0.1 M CHES, pH 8.9-9.9, X-ray diffraction structure determination and analysis at 1.8 A and 2.0-2.9 A resolution, respectively, molecular replacement method using the structure of FabG from Escherichia coli, PDB ID 1I01, as the search probe | Cupriavidus necator |
Protein Variants | Comment | Organism |
---|---|---|
additional information | directed evolution and structural analysis of NADPH-dependent acetoacetyl-CoA reductase reveals two mutations responsible for enhanced kinetics, enzyme mutant is engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants reveals that the beneficial mutations affect the flexibility around the active site, which in turn play an important role in substrate recognition. Both the kinetic analysis and crystal structure data support the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA | Cupriavidus necator |
Q47L | random mutagenesis, the mutant exhibits a kcat value 2.4fold higher compared to the wild-type enzyme, enhanced activity, and enhanced P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. The mutation affects the interaction with substrates, resulting in the acquirement of enhanced activity | Cupriavidus necator |
T173S | random mutagenesis, the mutant exhibits a kcat value 3.5fold higher compared to the wild-type enzyme, enhanced activity, and enhanced P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. The mutation affects the interaction with substrates, resulting in the acquirement of enhanced activity | Cupriavidus necator |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-3-hydroxyacyl-CoA + NADP+ | Cupriavidus necator | - |
3-oxoacyl-CoA + NADPH + H+ | - |
r | |
(R)-3-hydroxyacyl-CoA + NADP+ | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | - |
3-oxoacyl-CoA + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Cupriavidus necator | P14697 | - |
- |
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | P14697 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Cupriavidus necator |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-3-hydroxyacyl-CoA + NADP+ | - |
Cupriavidus necator | 3-oxoacyl-CoA + NADPH + H+ | - |
r | |
(R)-3-hydroxyacyl-CoA + NADP+ | - |
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | 3-oxoacyl-CoA + NADPH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
tetramer | - |
Cupriavidus necator |
Synonyms | Comment | Organism |
---|---|---|
acetoacetyl-CoA reductase | - |
Cupriavidus necator |
NADPH-dependent acetoacetyl coenzyme A reductase | - |
Cupriavidus necator |
PhaB | - |
Cupriavidus necator |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Cupriavidus necator |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Cupriavidus necator |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | dependent on | Cupriavidus necator | |
NADPH | - |
Cupriavidus necator |
General Information | Comment | Organism |
---|---|---|
additional information | PhaB forms a ternary complex with NADPH and acetoacetyl-CoA | Cupriavidus necator |