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Literature summary extracted from

  • Rakus, J.F.; Kalyanaraman, C.; Fedorov, A.A.; Fedorov, E.V.; Mills-Groninger, F.P.; Toro, R.; Bonanno, J.; Bain, K.; Sauder, J.M.; Burley, S.K.; Almo, S.C.; Jacobson, M.P.; Gerlt, J.A.
    Computation-facilitated assignment of the function in the enolase superfamily: a regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis (2009), Biochemistry, 48, 11546-11558.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Crystallization (Commentary)

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

Protein Variants

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

KM Value [mM]

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

Metals/Ions

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

Organism

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
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.42 by centrifugation, sonication, on Ni-NTA column and by gel filtration Oceanobacillus iheyensis

Substrates and Products (Substrate)

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 ?

Synonyms

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

Turnover Number [1/s]

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

kcat/KM [mM/s]

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