The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

Sugar interaction with metal ions. Crystal structure and FT-IR spectroscopic study of strontium galactarate mono-hydrate

The crystal structure of strontium galactarate mono-hydrate, Sr2+ x C6H8O8(2-) x H2O, Mr = 313.76, monoclinic, P2(1)/c, a = 10.268(2), b = 10.333(2), c = 10.194(2) A, beta = 117.87(3) degrees, lambda(Mo K alpha) = 0.71073 A, Z = 4, Dx = 2.180 Mg m(-3), V = 956.1(3) A3, mu = 5.676 mm(-1), F(000) = 624, T = 293(2) K, R = 0.0260 for 1690 observed reflections and 145 parameters refined, has been determined. The galactarate ion is centro-symmetrical in the crystal structure, although it contains independent half-ions. The Sr2+ ion is nine-coordinated (tricapped trigonal prism) with five Sr-O bonds from carboxylic groups, and four from hydroxyl groups. The water molecule does not take part in the coordination. Six hydrogen bonds are formed, three of them related to the water molecule. The spectroscopic evidence shows that the carboxylic acid dimers of the free acid dissociate. The asymmetric stretching vibrations of the anionic COO groups in the salt are observed at 1609 and 1548, and 1581 cm(-1), assigned to a mono-dentate and a tetra-dentate coordination, respectively. The symmetric stretching vibration is located at 1397 cm(-1). The hydroxyl groups of the galactarate skeleton take part in the metal-oxygen interaction, and the hydrogen-bonding network is rearranged upon sugar metalation.     

Structure of galactarate dehydratase,a new fold in an enolase involved in bacterial fitness after antibiotic treatment

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3‐keto‐5‐dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic‐treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N‐terminal domain was determined previously. Herein, we report the first crystal structure of full‐length GarD solved using a seleno‐methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N‐terminal domain is comprised of a β‐clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C‐terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven‐stranded parallel β‐sheet supported by nine α‐helices. A metal binding site in the C‐terminal domain is occupied by Ca²⁺. The activity of GarD was corroborated by the production of 5‐keto‐4‐deoxy‐D‐glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.     

Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2

We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. 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 alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 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 His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.