title: Structure and function of a decarboxylating Agrobacterium tumefaciens keto-deoxy-d-galactarate dehydratase.
abstract: Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.
authors.affiliation: Department of Chemistry, University of Eastern Finland , FI-80101 Joensuu, Finland.
authors.forename: Helena
authors.initials: H
authors.lastname: Taberman
authors.forename: Martina
authors.initials: M
authors.lastname: Andberg
authors.forename: Tarja
authors.initials: T
authors.lastname: Parkkinen
authors.forename: Janne
authors.initials: J
authors.lastname: Jänis
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.forename: Nina
authors.initials: N
authors.lastname: Hakulinen
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
authors.forename: Juha
authors.initials: J
authors.lastname: Rouvinen
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013400:Sugar Acids; C000090:galactaric acid; D000446:Aldehyde-Lyases; C021468:2-keto-3-deoxy-D-glucarate aldolase; D006836:Hydro-Lyases