Literature summary for 1.1.1.36 extracted from

Matsumoto, K.; Tanaka, Y.; Watanabe, T.; Motohashi, R.; Ikeda, K.; Tobitani, K.; Yao, M.; Tanaka, I.; Taguchi, S.
Directed evolution and structural analysis of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics (2013), Appl. Environ. Microbiol., 79, 6134-6139.

Cloned(Commentary)

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 (Commentary)

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

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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Cupriavidus necator	P14697	-	-
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1	P14697	-	-

Purification (Commentary)

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 and Products (Substrate)

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

Subunits	Comment	Organism
tetramer	-	Cupriavidus necator

Synonyms

Synonyms	Comment	Organism
acetoacetyl-CoA reductase	-	Cupriavidus necator
NADPH-dependent acetoacetyl coenzyme A reductase	-	Cupriavidus necator
PhaB	-	Cupriavidus necator

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Cupriavidus necator

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Cupriavidus necator

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	dependent on	Cupriavidus necator
NADPH	-	Cupriavidus necator

General Information

General Information	Comment	Organism
additional information	PhaB forms a ternary complex with NADPH and acetoacetyl-CoA	Cupriavidus necator