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Literature summary 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.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.36 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
1.1.1.100 expressed in Escherichia coli BL21(DE3) cells and in Corynebacterium glutamicum Cupriavidus necator

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.36 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
1.1.1.100 vapor diffusion method, using 0.1 M MES (pH 7.1), 1.6 M ammonium sulfate, and 10% (w/v) 1,4-dioxane Cupriavidus necator

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.36 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
1.1.1.36 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
1.1.1.36 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
1.1.1.100 Q47L the mutant with increased specific activity exhibits a kcat value that is 2.4fold higher than that of the wild type enzyme Cupriavidus necator
1.1.1.100 T173S the mutant with increased specific activity exhibits a kcat value that is 3.5fold higher than that of the wild type enzyme Cupriavidus necator

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.100 0.0057
-
 acetoacetyl-CoA wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 0.0136
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 0.0159
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 0.149
-
 NADPH wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 0.289
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 0.617
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.36 (R)-3-hydroxyacyl-CoA + NADP+ Cupriavidus necator
-
 3-oxoacyl-CoA + NADPH + H+
-
 r
1.1.1.36 (R)-3-hydroxyacyl-CoA + NADP+ Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
 3-oxoacyl-CoA + NADPH + H+
-
 r
1.1.1.100 acetoacetyl-CoA + NADPH + H+ Cupriavidus necator
-
 3-hydroxybutyryl-CoA + NADP+
-
 ?
1.1.1.100 acetoacetyl-CoA + NADPH + H+ Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
 3-hydroxybutyryl-CoA + NADP+
-
 ?

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.36 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Cupriavidus necator
1.1.1.100 His-Bind resin column chromatography and Superdex 200 pg gel filtration Cupriavidus necator

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.36 (R)-3-hydroxyacyl-CoA + NADP+
-
 Cupriavidus necator 3-oxoacyl-CoA + NADPH + H+
-
 r
1.1.1.36 (R)-3-hydroxyacyl-CoA + NADP+
-
 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 3-oxoacyl-CoA + NADPH + H+
-
 r
1.1.1.100 acetoacetyl-CoA + NADPH + H+
-
 Cupriavidus necator 3-hydroxybutyryl-CoA + NADP+
-
 ?
1.1.1.100 acetoacetyl-CoA + NADPH + H+
-
 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 3-hydroxybutyryl-CoA + NADP+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.1.1.36 tetramer
-
 Cupriavidus necator

Synonyms

EC Number Synonyms Comment Organism
1.1.1.36 acetoacetyl-CoA reductase
-
 Cupriavidus necator
1.1.1.36 NADPH-dependent acetoacetyl coenzyme A reductase
-
 Cupriavidus necator
1.1.1.36 PhaB
-
 Cupriavidus necator
1.1.1.100 NADPH-dependent acetoacetyl coenzyme A reductase
-
 Cupriavidus necator
1.1.1.100 PhaB
-
 Cupriavidus necator

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.36 30
-
 assay at Cupriavidus necator

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.100 102
-
 NADPH wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 102
-
 acetoacetyl-CoA wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 249
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 249
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 361
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 361
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.36 8
-
 assay at Cupriavidus necator

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.36 NADP+ dependent on Cupriavidus necator
1.1.1.36 NADPH
-
 Cupriavidus necator
1.1.1.100 NADP+
-
 Cupriavidus necator

General Information

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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.100 585
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 685
-
 NADPH wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 862
-
 NADPH mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 15200
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 18000
-
 acetoacetyl-CoA wild type enzyme, at pH 8.0 and 30°C Cupriavidus necator
1.1.1.100 26500
-
 acetoacetyl-CoA mutant enzyme Q47L, at pH 8.0 and 30°C Cupriavidus necator