PMID:22493433

Citation	Andberg, M, Maaheimo, H, Boer, H, Penttilä, M, Koivula, A and Richard, P (2012) Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate. J. Biol. Chem. 287:17662-71
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.
Links	PubMed PMC3366811 Online version:10.1074/jbc.M111.335240
Keywords	Agrobacterium tumefaciens/enzymology; Agrobacterium tumefaciens/genetics; Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Intramolecular Lyases/genetics; Intramolecular Lyases/metabolism; Lactones/metabolism; Molecular Sequence Data
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
AGRT5:A9CEQ8		GO:0019698: D-galacturonate catabolic process	ECO:0000314:		P		Fig 2. SDS-PAGE of purified Gci. Fig 4 shows the signals of the HNMR spectrum of a complete reaction mixture of galactaric acid and D-galactaro-1,4-lactone with Gci identical to the reaction using the control enzyme.	complete
AGRFC:GCI	Contributes to	GO:0009063: cellular amino acid catabolic process	ECO:0000314:		P		Different pathways for d-galacturonate catabolism take place. It is active both on d-galactarolactone and d-glucarolactone, but does not work on the corresponding linear hexaric acid forms. These verifications are confirmed through the use of a gel (Fig. 2). Figure 1 shows the reaction scheme of the first steps of the oxidation pathways, which further clarify the function.	complete

References

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