title: Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate.
abstract: Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland. Electronic address: martina.andberg@vtt.fi.
authors.forename: Martina
authors.initials: M
authors.lastname: Andberg
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Hannu
authors.initials: H
authors.lastname: Maaheimo
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Harry
authors.initials: H
authors.lastname: Boer
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Peter
authors.initials: P
authors.lastname: Richard
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D007783:Lactones; D019753:Intramolecular Lyases