The enzyme from the bacterium Escherichia coli is specific for galactarate , while the enzyme from Salmonella typhimurium also has activity with L-talarate (cf. EC 4.2.1.156, L-talarate dehydratase) . cf. EC 4.2.1.158, galactarate dehydratase (D-threo-forming).
The enzyme from the bacterium Escherichia coli is specific for galactarate [2], while the enzyme from Salmonella typhimurium also has activity with L-talarate (cf. EC 4.2.1.156, L-talarate dehydratase) [3]. cf. EC 4.2.1.158, galactarate dehydratase (D-threo-forming).
galactarate i.e. (2R,3S,4R,5S)-2,3,4,5-tetrahydroxyhexanedioate. The enzyme also catalyzes dehydration of L-talarate. The dehydration of L-talarate is accompanied by competing epimerization to galactarate. Little epimerization to L-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with L-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with L-glucarate, a substrate for exchange of the R-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, it is concluded that Lys197 functions as the galactarate-specific base and His328 functions as the L-talarate-specific base. The epimerization of L-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His328) and epimerization (protonation on C2 by the conjugate acid of Lys197). Only galactarate and L-talarate are completely dehydrated by the enzyme. No other acid sugar results in detectable turnover
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
disruption of gene STM3697 diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. Disruption eliminates the ability to grow on L-talarate
disruption of gene STM3697 diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. Disruption eliminates the ability to grow on L-talarate
structures of the wild type enzyme complexed with L-lyxarohydroxamate, and of the K197A mutant complexed with L-glucarate. Residue Lys 197 functions as the galactarate-specific base and His 328 functions as the L-talarate-specific base
three different crystal forms are grown by hanging drop vapor diffusion at room temperature: (1) selenomethionine (SeMet)-substituted wild type enzyme, (2) wild-type enzyme complexed with Mg2+ and L-lyxarohydroxamate, and (3) the K197A mutant enzyme complexed with Mg2+ and L-glucarate
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
inactive mutant enzyme, the structure of the K197A mutant enzyme complexed with Mg2+ and L-glucarate is determined by molecular replacement using the SeMet-substituted STM3697 structure as the search model
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
a continuous assay for L-talarate/galactarate dehydratase is develped using circular dichroism. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes
a convenient, direct continuous circular dichroism-based assay is developed for following the L-talarate/galactarate dehydratase-catalyzed conversion of meso-galactarate to 5-dehydro-4-deoxy-D-glucarate. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes
a continuous assay for L-talarate/galactarate dehydratase is develped using circular dichroism. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes
a convenient, direct continuous circular dichroism-based assay is developed for following the L-talarate/galactarate dehydratase-catalyzed conversion of meso-galactarate to 5-dehydro-4-deoxy-D-glucarate. The advantages that this assay method offers are that initial rate measurements may be obtained much more rapidly than is possible using fixed-time assays (e.g., semicarbazide derivatization or NMR methods) and it is much less complex than a coupled assay, which is often limited by the availability and specific properties of the coupling enzymes
Computation-facilitated assignment of the function in the enolase superfamily: a regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis