EC Number | Cloned (Comment) | Organism |
---|---|---|
4.2.1.42 | into a TOPO vector (pSGX3), and transformed into Escherichia coli TOP10 competent cells. Gene in the pSGX3 vector transformed into Escherichia coli strains XL1-Blue for transformation and BL21(DE3) for expression | Oceanobacillus iheyensis |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
4.2.1.158 | wild-type and mutant Y90F. A Tyr 164-Arg 162 dyad is the base that initiates the reaction by abstraction of the alpha-proton and Tyr 90 is the acid that facilitates departure of the beta-OH leaving group. The structure contains two Mg2+ ions located 10.4 A from one another, with one located in the canonical position in the (beta/alpha)7beta-barrel, the second is located in a site within the capping domain | Oceanobacillus iheyensis |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
4.2.1.42 | H45Q | has no detectable activity | Oceanobacillus iheyensis |
4.2.1.42 | R162N | retains a small amount of activity | Oceanobacillus iheyensis |
4.2.1.42 | Y164F | has no detectable activity | Oceanobacillus iheyensis |
4.2.1.42 | Y90F | is catalytically impaired. Structure of the mutant in complex with Mg2+ and galactarate has a well-defined C-terminal segment through residue 387, well-ordered electron density for galactarate, and Mg2+ ions in both metal sites for both protomers comprising the asymmetric unit | Oceanobacillus iheyensis |
4.2.1.158 | Y90F | catalytically impaired | Oceanobacillus iheyensis |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.42 | 0.62 | - |
galactarate | wild-type, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 2 | - |
galactarate | mutant Y90F, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 4.4 | - |
galactarate | mutant H45Q, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.158 | 0.62 | - |
galactarate | pH 8.0, 30°C | Oceanobacillus iheyensis |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
4.2.1.42 | Mg2+ | structure contains two Mg2+ ions located 10.4 A from one another, with one located in the canonical position in the (beta/alpha)7beta-barrel domain, the second is located in a site within the capping domain | Oceanobacillus iheyensis | |
4.2.1.158 | Mg2+ | structure contains two Mg2+ ions | Oceanobacillus iheyensis |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
4.2.1.42 | Oceanobacillus iheyensis | - |
- |
- |
4.2.1.158 | Oceanobacillus iheyensis | Q8EMJ9 | - |
- |
4.2.1.158 | Oceanobacillus iheyensis DSM 14371 | Q8EMJ9 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
4.2.1.42 | by centrifugation, sonication, on Ni-NTA column and by gel filtration | Oceanobacillus iheyensis |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
4.2.1.42 | galactarate | - |
Oceanobacillus iheyensis | 5-dehydro-4-deoxy D-glucarate + H2O | - |
? | |
4.2.1.158 | galactarate | - |
Oceanobacillus iheyensis | 2-oxo-D-threo-4,5-dihydroxyadipate + H2O | i.e. (2S,3R)-2,3-dihydroxy-5-oxohexanedioate. Product is the enantiomer of the product obtained in the reaction catalyzed by bifunctional L-talarate/galactarate dehydratase. When the GalrD-II-catalyzed reaction is performed in D2O, the proS hydrogen is replaced with deuterium, i.e., the acid that facilitates the departure of the OH group is likely the acid that catalyzes the formation of the alpha-oxo-beta-methylene product | ? | |
4.2.1.158 | galactarate | - |
Oceanobacillus iheyensis DSM 14371 | 2-oxo-D-threo-4,5-dihydroxyadipate + H2O | i.e. (2S,3R)-2,3-dihydroxy-5-oxohexanedioate. Product is the enantiomer of the product obtained in the reaction catalyzed by bifunctional L-talarate/galactarate dehydratase. When the GalrD-II-catalyzed reaction is performed in D2O, the proS hydrogen is replaced with deuterium, i.e., the acid that facilitates the departure of the OH group is likely the acid that catalyzes the formation of the alpha-oxo-beta-methylene product | ? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
4.2.1.42 | galactarate dehydratase | - |
Oceanobacillus iheyensis |
4.2.1.42 | GalrD | - |
Oceanobacillus iheyensis |
4.2.1.158 | galactarate dehydratase (D-threo-forming) | - |
Oceanobacillus iheyensis |
4.2.1.158 | GalrD-II | - |
Oceanobacillus iheyensis |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.42 | 0.0013 | - |
galactarate | mutant H45Q, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 0.0061 | - |
galactarate | mutant Y90F, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 6.8 | - |
galactarate | wild-type, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.158 | 6.8 | - |
galactarate | pH 8.0, 30°C | Oceanobacillus iheyensis |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
4.2.1.42 | 0.00029 | - |
galactarate | mutant H45Q, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 0.00065 | - |
galactarate | mutant R162N, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 0.0031 | - |
galactarate | mutant Y90F, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.42 | 11 | - |
galactarate | wild-type, in 50 mM Hepes, pH 8.0, 5 mM MgCl2 | Oceanobacillus iheyensis | |
4.2.1.158 | 11 | - |
galactarate | pH 8.0, 30°C | Oceanobacillus iheyensis |