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Literature summary extracted from

  • Nakashima, K.; Ito, K.; Nakajima, Y.; Yamazawa, R.; Miyakawa, S.; Yoshimoto, T.
    Closed complex of the D-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor (2009), J. Biochem., 145, 467-479.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.30 wild-type and mutants expressed in Escherichia coli XL1Blue harbouring pHBDH11. Wild-type HBDH and mutants containing an N-terminal His-tag expressed in Escherichia coli XL1Blue using pQE30 as a vector Pseudomonas fragi

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.30 crystals of ternary complex of HBDH-NAD+-L-3-hydroxybutyrate and the binary complex of HBDH-NAD+. The former structure shows a closed-form conformation, which is considered an active form for catalysis, while the latter stays mostly in a open-form conformation. Crystals of mutants T190S and T190A Pseudomonas fragi

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.30 H144A catalytic efficiency (kcat/Km) is 0.2% of the activity of wild-type HBDH Pseudomonas fragi
1.1.1.30 K152A no activity Pseudomonas fragi
1.1.1.30 K152E very low activity Pseudomonas fragi
1.1.1.30 K152Q very low activity Pseudomonas fragi
1.1.1.30 K152R retains a significant level of activity Pseudomonas fragi
1.1.1.30 L215A both Km and kcat values are largely affected and the catalytic efficiency (kcat/Km) is less than 3% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 L215V Km values increase 3.5- and 4.3fold and the kcat values are 73-118% those of the wild-type toward D-3-hydroxybutyrate and acetoacetate, respectively. Mutation does not significantly change Km and kcat toward NAD+ and NADH Pseudomonas fragi
1.1.1.30 Q196A kcat/Km value is 0.6% that of the wild-type Pseudomonas fragi
1.1.1.30 Q196E substantially reduced activity Pseudomonas fragi
1.1.1.30 Q196N substantially reduced activity Pseudomonas fragi
1.1.1.30 Q94A catalytic efficiency (kcat/Km) is 1.4% of the activity of wild-type HBDH Pseudomonas fragi
1.1.1.30 T190A activity decreases to 0.1% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 T190C decreased activity Pseudomonas fragi
1.1.1.30 T190S retains 37% of the activity Pseudomonas fragi
1.1.1.30 W187A very low activity Pseudomonas fragi
1.1.1.30 W187F shows significant activity levels, 65% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 W187T shows faint activity Pseudomonas fragi
1.1.1.30 W187Y shows significant activity levels, 41% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 W257A no activity Pseudomonas fragi
1.1.1.30 W257F shows low activity levels, 2% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 W257Y shows low activity levels, 1% that of the wild-type enzyme Pseudomonas fragi
1.1.1.30 Y155F no activity Pseudomonas fragi

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.30 L-3-hydroxybutyrate is a competitive inhibitor Pseudomonas fragi

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.30 0.01
-
 NADH wild-type Pseudomonas fragi
1.1.1.30 0.02
-
 NADH mutant T190S Pseudomonas fragi
1.1.1.30 0.03
-
 NADH mutant L215V Pseudomonas fragi
1.1.1.30 0.1
-
 NADH mutant T190A Pseudomonas fragi
1.1.1.30 0.12
-
 NAD+ mutant H6-K152R Pseudomonas fragi
1.1.1.30 0.12
-
 NAD+ mutant H6-Q196A Pseudomonas fragi
1.1.1.30 0.12
-
 NAD+ mutant H6-Q196E Pseudomonas fragi
1.1.1.30 0.17
-
 NAD+ H6-HBDH Pseudomonas fragi
1.1.1.30 0.19
-
 NAD+ mutant H6-Q94A Pseudomonas fragi
1.1.1.30 0.2
-
 NAD+ mutant H6-Q196N Pseudomonas fragi
1.1.1.30 0.21
-
 NAD+ mutant H6-W187Y Pseudomonas fragi
1.1.1.30 0.24
-
 NAD+ wild-type Pseudomonas fragi
1.1.1.30 0.24
-
 NAD+ mutant H6-W187F Pseudomonas fragi
1.1.1.30 0.29
-
 NAD+ mutant L215V Pseudomonas fragi
1.1.1.30 0.35
-
 NAD+ mutant H6-H144A Pseudomonas fragi
1.1.1.30 0.37
-
 acetoacetate wild-type Pseudomonas fragi
1.1.1.30 0.49
-
 NAD+ mutant H6-W257F Pseudomonas fragi
1.1.1.30 0.5
-
 NADH mutant L215A Pseudomonas fragi
1.1.1.30 0.69
-
 NAD+ mutant T190C Pseudomonas fragi
1.1.1.30 0.71
-
 NAD+ mutant T190S Pseudomonas fragi
1.1.1.30 0.74
-
 NAD+ mutant H6-W257Y Pseudomonas fragi
1.1.1.30 0.76
-
 NAD+ mutant H6-W187A Pseudomonas fragi
1.1.1.30 0.8
-
 D-3-hydroxybutyrate wild-type Pseudomonas fragi
1.1.1.30 0.88
-
 D-3-hydroxybutyrate H6-HBDH Pseudomonas fragi
1.1.1.30 0.88
-
 NAD+ mutant H6-W187T Pseudomonas fragi
1.1.1.30 0.95
-
 NAD+ mutant H6-W257A Pseudomonas fragi
1.1.1.30 1.1
-
 acetoacetate mutant T190S Pseudomonas fragi
1.1.1.30 1.2
-
 D-3-hydroxybutyrate mutant H6-W187Y Pseudomonas fragi
1.1.1.30 1.3
-
 D-3-hydroxybutyrate mutant H6-W187F Pseudomonas fragi
1.1.1.30 1.3
-
 acetoacetate mutant L215V Pseudomonas fragi
1.1.1.30 1.5
-
 NAD+ mutant L215A Pseudomonas fragi
1.1.1.30 1.5
-
 NADH mutant T190C Pseudomonas fragi
1.1.1.30 2.2
-
 NAD+ mutant T190A Pseudomonas fragi
1.1.1.30 3.4
-
 D-3-hydroxybutyrate mutant L215V Pseudomonas fragi
1.1.1.30 3.7
-
 D-3-hydroxybutyrate mutant T190S Pseudomonas fragi
1.1.1.30 7.7
-
 acetoacetate mutant T190C Pseudomonas fragi
1.1.1.30 8.3
-
 acetoacetate mutant L215A Pseudomonas fragi
1.1.1.30 9.2
-
 D-3-hydroxybutyrate mutant L215A Pseudomonas fragi
1.1.1.30 25
-
 D-3-hydroxybutyrate mutant H6-H144A Pseudomonas fragi
1.1.1.30 25
-
 D-3-hydroxybutyrate mutant T190A Pseudomonas fragi
1.1.1.30 30
-
 D-3-hydroxybutyrate mutant H6-Q94A Pseudomonas fragi
1.1.1.30 31
-
 D-3-hydroxybutyrate mutant H6-K152R Pseudomonas fragi
1.1.1.30 32
-
 acetoacetate mutant T190A Pseudomonas fragi
1.1.1.30 36
-
 D-3-hydroxybutyrate mutant T190C Pseudomonas fragi
1.1.1.30 47
-
 D-3-hydroxybutyrate mutant H6-Q196A Pseudomonas fragi
1.1.1.30 51
-
 D-3-hydroxybutyrate mutant H6-Q196N Pseudomonas fragi
1.1.1.30 61
-
 D-3-hydroxybutyrate mutant H6-W257A Pseudomonas fragi
1.1.1.30 65
-
 D-3-hydroxybutyrate mutant H6-W257Y Pseudomonas fragi
1.1.1.30 83
-
 D-3-hydroxybutyrate mutant H6-Q196E Pseudomonas fragi
1.1.1.30 83
-
 D-3-hydroxybutyrate mutant H6-W257F Pseudomonas fragi
1.1.1.30 84
-
 D-3-hydroxybutyrate mutant H6-W187T Pseudomonas fragi
1.1.1.30 100
-
 D-3-hydroxybutyrate mutant H6-W187A Pseudomonas fragi

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.30 Pseudomonas fragi Q5KST5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.30 wild-type and mutants purified by metal chelating affinity chromatography followed by ion-exchange chromatography Pseudomonas fragi

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.30 D-3-hydroxybutyrate + NAD+
-
 Pseudomonas fragi acetoacetate + NADH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.1.1.30 HBDH
-
 Pseudomonas fragi

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.30 4.5
-
 acetoacetate mutant T190A Pseudomonas fragi
1.1.1.30 5.1
-
 acetoacetate mutant T190C Pseudomonas fragi
1.1.1.30 6.1
-
 D-3-hydroxybutyrate mutant T190C Pseudomonas fragi
1.1.1.30 6.7
-
 D-3-hydroxybutyrate mutant H6-W257A Pseudomonas fragi
1.1.1.30 7.2
-
 D-3-hydroxybutyrate mutant H6-W187T Pseudomonas fragi
1.1.1.30 12
-
 D-3-hydroxybutyrate mutant H6-W257Y Pseudomonas fragi
1.1.1.30 21
-
 D-3-hydroxybutyrate mutant H6-H144A Pseudomonas fragi
1.1.1.30 22
-
 D-3-hydroxybutyrate mutant H6-Q196N Pseudomonas fragi
1.1.1.30 22
-
 D-3-hydroxybutyrate mutant T190A Pseudomonas fragi
1.1.1.30 29
-
 D-3-hydroxybutyrate mutant H6-W187A Pseudomonas fragi
1.1.1.30 30
-
 acetoacetate mutant L215A Pseudomonas fragi
1.1.1.30 70
-
 D-3-hydroxybutyrate mutant L215A Pseudomonas fragi
1.1.1.30 83
-
 D-3-hydroxybutyrate mutant H6-Q196E Pseudomonas fragi
1.1.1.30 87
-
 acetoacetate mutant L215V Pseudomonas fragi
1.1.1.30 88
-
 D-3-hydroxybutyrate mutant H6-W257F Pseudomonas fragi
1.1.1.30 99
-
 D-3-hydroxybutyrate mutant H6-Q196A Pseudomonas fragi
1.1.1.30 110
-
 D-3-hydroxybutyrate mutant H6-K152R Pseudomonas fragi
1.1.1.30 120
-
 acetoacetate wild-type Pseudomonas fragi
1.1.1.30 125
-
 acetoacetate mutant T190S Pseudomonas fragi
1.1.1.30 140
-
 D-3-hydroxybutyrate mutant H6-Q94A Pseudomonas fragi
1.1.1.30 180
-
 D-3-hydroxybutyrate mutant H6-W187Y Pseudomonas fragi
1.1.1.30 310
-
 D-3-hydroxybutyrate H6-HBDH Pseudomonas fragi
1.1.1.30 310
-
 D-3-hydroxybutyrate mutant H6-W187F Pseudomonas fragi
1.1.1.30 370
-
 D-3-hydroxybutyrate wild-type Pseudomonas fragi
1.1.1.30 435
-
 D-3-hydroxybutyrate mutant L215V Pseudomonas fragi
1.1.1.30 705
-
 D-3-hydroxybutyrate mutant T190S Pseudomonas fragi

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.30 8.5
-
-
 Pseudomonas fragi