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Structure of galactarate dehydratase,a new fold in an enolase involved in bacterial fitness after antibiotic treatment
Authors:Monica Rosas‐Lemus  George Minasov  Ludmilla Shuvalova  Zdzislaw Wawrzak  Olga Kiryukhina  Nathan Mih  Lukasz Jaroszewski  Bernhard Palsson  Adam Godzik  Karla J F Satchell
Abstract: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.
Keywords:antibiotic treatment  bacterial fitness  enolase     Escherichia coli  galactarate dehydratase  intestinal pathogens  novel fold
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