Cloned (Comment) | Organism |
---|---|
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 (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
H144A | catalytic efficiency (kcat/Km) is 0.2% of the activity of wild-type HBDH | Pseudomonas fragi |
K152A | no activity | Pseudomonas fragi |
K152E | very low activity | Pseudomonas fragi |
K152Q | very low activity | Pseudomonas fragi |
K152R | retains a significant level of activity | Pseudomonas fragi |
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 |
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 |
Q196A | kcat/Km value is 0.6% that of the wild-type | Pseudomonas fragi |
Q196E | substantially reduced activity | Pseudomonas fragi |
Q196N | substantially reduced activity | Pseudomonas fragi |
Q94A | catalytic efficiency (kcat/Km) is 1.4% of the activity of wild-type HBDH | Pseudomonas fragi |
T190A | activity decreases to 0.1% that of the wild-type enzyme | Pseudomonas fragi |
T190C | decreased activity | Pseudomonas fragi |
T190S | retains 37% of the activity | Pseudomonas fragi |
W187A | very low activity | Pseudomonas fragi |
W187F | shows significant activity levels, 65% that of the wild-type enzyme | Pseudomonas fragi |
W187T | shows faint activity | Pseudomonas fragi |
W187Y | shows significant activity levels, 41% that of the wild-type enzyme | Pseudomonas fragi |
W257A | no activity | Pseudomonas fragi |
W257F | shows low activity levels, 2% that of the wild-type enzyme | Pseudomonas fragi |
W257Y | shows low activity levels, 1% that of the wild-type enzyme | Pseudomonas fragi |
Y155F | no activity | Pseudomonas fragi |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
L-3-hydroxybutyrate | is a competitive inhibitor | Pseudomonas fragi |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
NADH | wild-type | Pseudomonas fragi | |
0.02 | - |
NADH | mutant T190S | Pseudomonas fragi | |
0.03 | - |
NADH | mutant L215V | Pseudomonas fragi | |
0.1 | - |
NADH | mutant T190A | Pseudomonas fragi | |
0.12 | - |
NAD+ | mutant H6-K152R | Pseudomonas fragi | |
0.12 | - |
NAD+ | mutant H6-Q196A | Pseudomonas fragi | |
0.12 | - |
NAD+ | mutant H6-Q196E | Pseudomonas fragi | |
0.17 | - |
NAD+ | H6-HBDH | Pseudomonas fragi | |
0.19 | - |
NAD+ | mutant H6-Q94A | Pseudomonas fragi | |
0.2 | - |
NAD+ | mutant H6-Q196N | Pseudomonas fragi | |
0.21 | - |
NAD+ | mutant H6-W187Y | Pseudomonas fragi | |
0.24 | - |
NAD+ | wild-type | Pseudomonas fragi | |
0.24 | - |
NAD+ | mutant H6-W187F | Pseudomonas fragi | |
0.29 | - |
NAD+ | mutant L215V | Pseudomonas fragi | |
0.35 | - |
NAD+ | mutant H6-H144A | Pseudomonas fragi | |
0.37 | - |
acetoacetate | wild-type | Pseudomonas fragi | |
0.49 | - |
NAD+ | mutant H6-W257F | Pseudomonas fragi | |
0.5 | - |
NADH | mutant L215A | Pseudomonas fragi | |
0.69 | - |
NAD+ | mutant T190C | Pseudomonas fragi | |
0.71 | - |
NAD+ | mutant T190S | Pseudomonas fragi | |
0.74 | - |
NAD+ | mutant H6-W257Y | Pseudomonas fragi | |
0.76 | - |
NAD+ | mutant H6-W187A | Pseudomonas fragi | |
0.8 | - |
D-3-hydroxybutyrate | wild-type | Pseudomonas fragi | |
0.88 | - |
D-3-hydroxybutyrate | H6-HBDH | Pseudomonas fragi | |
0.88 | - |
NAD+ | mutant H6-W187T | Pseudomonas fragi | |
0.95 | - |
NAD+ | mutant H6-W257A | Pseudomonas fragi | |
1.1 | - |
acetoacetate | mutant T190S | Pseudomonas fragi | |
1.2 | - |
D-3-hydroxybutyrate | mutant H6-W187Y | Pseudomonas fragi | |
1.3 | - |
D-3-hydroxybutyrate | mutant H6-W187F | Pseudomonas fragi | |
1.3 | - |
acetoacetate | mutant L215V | Pseudomonas fragi | |
1.5 | - |
NAD+ | mutant L215A | Pseudomonas fragi | |
1.5 | - |
NADH | mutant T190C | Pseudomonas fragi | |
2.2 | - |
NAD+ | mutant T190A | Pseudomonas fragi | |
3.4 | - |
D-3-hydroxybutyrate | mutant L215V | Pseudomonas fragi | |
3.7 | - |
D-3-hydroxybutyrate | mutant T190S | Pseudomonas fragi | |
7.7 | - |
acetoacetate | mutant T190C | Pseudomonas fragi | |
8.3 | - |
acetoacetate | mutant L215A | Pseudomonas fragi | |
9.2 | - |
D-3-hydroxybutyrate | mutant L215A | Pseudomonas fragi | |
25 | - |
D-3-hydroxybutyrate | mutant H6-H144A | Pseudomonas fragi | |
25 | - |
D-3-hydroxybutyrate | mutant T190A | Pseudomonas fragi | |
30 | - |
D-3-hydroxybutyrate | mutant H6-Q94A | Pseudomonas fragi | |
31 | - |
D-3-hydroxybutyrate | mutant H6-K152R | Pseudomonas fragi | |
32 | - |
acetoacetate | mutant T190A | Pseudomonas fragi | |
36 | - |
D-3-hydroxybutyrate | mutant T190C | Pseudomonas fragi | |
47 | - |
D-3-hydroxybutyrate | mutant H6-Q196A | Pseudomonas fragi | |
51 | - |
D-3-hydroxybutyrate | mutant H6-Q196N | Pseudomonas fragi | |
61 | - |
D-3-hydroxybutyrate | mutant H6-W257A | Pseudomonas fragi | |
65 | - |
D-3-hydroxybutyrate | mutant H6-W257Y | Pseudomonas fragi | |
83 | - |
D-3-hydroxybutyrate | mutant H6-Q196E | Pseudomonas fragi | |
83 | - |
D-3-hydroxybutyrate | mutant H6-W257F | Pseudomonas fragi | |
84 | - |
D-3-hydroxybutyrate | mutant H6-W187T | Pseudomonas fragi | |
100 | - |
D-3-hydroxybutyrate | mutant H6-W187A | Pseudomonas fragi |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas fragi | Q5KST5 | - |
- |
Purification (Comment) | Organism |
---|---|
wild-type and mutants purified by metal chelating affinity chromatography followed by ion-exchange chromatography | Pseudomonas fragi |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-3-hydroxybutyrate + NAD+ | - |
Pseudomonas fragi | acetoacetate + NADH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
HBDH | - |
Pseudomonas fragi |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
4.5 | - |
acetoacetate | mutant T190A | Pseudomonas fragi | |
5.1 | - |
acetoacetate | mutant T190C | Pseudomonas fragi | |
6.1 | - |
D-3-hydroxybutyrate | mutant T190C | Pseudomonas fragi | |
6.7 | - |
D-3-hydroxybutyrate | mutant H6-W257A | Pseudomonas fragi | |
7.2 | - |
D-3-hydroxybutyrate | mutant H6-W187T | Pseudomonas fragi | |
12 | - |
D-3-hydroxybutyrate | mutant H6-W257Y | Pseudomonas fragi | |
21 | - |
D-3-hydroxybutyrate | mutant H6-H144A | Pseudomonas fragi | |
22 | - |
D-3-hydroxybutyrate | mutant H6-Q196N | Pseudomonas fragi | |
22 | - |
D-3-hydroxybutyrate | mutant T190A | Pseudomonas fragi | |
29 | - |
D-3-hydroxybutyrate | mutant H6-W187A | Pseudomonas fragi | |
30 | - |
acetoacetate | mutant L215A | Pseudomonas fragi | |
70 | - |
D-3-hydroxybutyrate | mutant L215A | Pseudomonas fragi | |
83 | - |
D-3-hydroxybutyrate | mutant H6-Q196E | Pseudomonas fragi | |
87 | - |
acetoacetate | mutant L215V | Pseudomonas fragi | |
88 | - |
D-3-hydroxybutyrate | mutant H6-W257F | Pseudomonas fragi | |
99 | - |
D-3-hydroxybutyrate | mutant H6-Q196A | Pseudomonas fragi | |
110 | - |
D-3-hydroxybutyrate | mutant H6-K152R | Pseudomonas fragi | |
120 | - |
acetoacetate | wild-type | Pseudomonas fragi | |
125 | - |
acetoacetate | mutant T190S | Pseudomonas fragi | |
140 | - |
D-3-hydroxybutyrate | mutant H6-Q94A | Pseudomonas fragi | |
180 | - |
D-3-hydroxybutyrate | mutant H6-W187Y | Pseudomonas fragi | |
310 | - |
D-3-hydroxybutyrate | H6-HBDH | Pseudomonas fragi | |
310 | - |
D-3-hydroxybutyrate | mutant H6-W187F | Pseudomonas fragi | |
370 | - |
D-3-hydroxybutyrate | wild-type | Pseudomonas fragi | |
435 | - |
D-3-hydroxybutyrate | mutant L215V | Pseudomonas fragi | |
705 | - |
D-3-hydroxybutyrate | mutant T190S | Pseudomonas fragi |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
- |
Pseudomonas fragi |