Cloned (Comment) | Organism |
---|---|
gene mdh, recombinant expression of C-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) | Thermus thermophilus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme in apoform and in complex with NAD+, sitting drop vapor diffusion method, mixing 500 nl of 10 mg/ml protein with an equal volume of mother liquor and equilibration against 0.1 ml of the crystallization solution containing 100 mM Tris-HCl, pH 8.2, 22.5% w/v PEG4000, and 200 mM magnesium chloride, at 10°C, 5 days. NAD-bound form crystals are obtained by soaking the apo-form crystal with a 001 ml crystallization reservoir containing 2 mM NAD+ molecule for about 30 min immediately prior X-ray diffraction, X-ray diffraction structure determination and analysis at 1.80-2.36 A resolution, molecular replacement and modelling, overview | Thermus thermophilus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
61000 | - |
recombinant enzyme, gel filtration | Thermus thermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-malate + NAD+ | Thermus thermophilus | - |
oxaloacetate + NADH + H+ | - |
r | |
oxaloacetate + NADH + H+ | Thermus thermophilus | - |
(S)-malate + NAD+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus thermophilus | P10584 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Thermus thermophilus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
2600 | - |
pH 10.0, 37°C | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-malate + NAD+ | - |
Thermus thermophilus | oxaloacetate + NADH + H+ | - |
r | |
additional information | crystal structures and molecular dynamics simulations of thermophilic malate dehydrogenase reveal critical loop motion for co-substrate binding | Thermus thermophilus | ? | - |
? | |
oxaloacetate + NADH + H+ | - |
Thermus thermophilus | (S)-malate + NAD+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 36874.5, recombinant enzyme, sequence calculation | Thermus thermophilus |
More | structure analysis, overview. Similar to other MDHs, a protomer of TtMDH has the N-terminal NAD binding domain and C-terminal catalytic domain and consists of 12 helices and 11 beta-strands. The N-terminal NAD binding domain (residue 1-156) is an open twisted structure with the classical Rossmann fold composed of a parallel six-stranded beta-sheet (beta1-beta6) and four alpha-helices (alpha1-alpha4). The C-terminal catalytic domain comprises an antiparallel twisted five-stranded sheet (beta7-beta11) surrounded by eight alpha-helices | Thermus thermophilus |
Synonyms | Comment | Organism |
---|---|---|
MDH | - |
Thermus thermophilus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
90 | - |
- |
Thermus thermophilus |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | 100 | high activity within this range | Thermus thermophilus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | 100 | high thermal stability of TtMDH, that does not reach a completely denatured state even at near 100°C | Thermus thermophilus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
- |
Thermus thermophilus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
8.5 | 11 | over 50% of maximal activity within this range | Thermus thermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | a slight movement of the enzymes binding loop and few structural elements around the co-substrate binding packet occurs in the presence of NAD+. The overall structures do not change much and retain all related positions | Thermus thermophilus |
General Information | Comment | Organism |
---|---|---|
additional information | molecular dynamics simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. At the simulated structure of 80°C, a large change occurs around the active site such that with increasing temperature, a mobile loop is closed to co-substrate binding region. The thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity | Thermus thermophilus |