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Galactaro δ-Lactone Isomerase: Lactone Isomerization by a Member of the Amidohydrolase Superfamily.

Bouvier JT, Groninger-Poe FP, Vetting M, Almo SC, Gerlt JA. Biochemistry 53, 614-6. PMCID: PMC3977579

In this publication, researchers from the EN Bridging Project collaborate with the Structure Core to identify the missing link in a pathway for D-galacturonate catabolism. In the process, they discover yet another function to add to the already extensive repertoire of the Amidohydrolase Superfamily. This research provides another successful example of an established EFI methodology: gene-neighborhood-driven functional discovery and subsequent pathway elucidation.

ABSTRACT

Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to d-galactaro-1,5-lactone. We have identified a novel enzyme, d-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of d-galactaro-1,5-lactone to d-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

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Abstract Image: Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to d-galactaro-1,5-lactone. We have identified a novel enzyme, d-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of d-galactaro-1,5-lactone to d-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

Figure 1: (A) d-Galacturonate oxidative catabolic pathway. (B) Gene cluster.

Figure 2: 1H NMR spectra of the isomerase substrate and product. (A) Synthetic δ-lactone immediately after the pD had been adjusted from 4.8 to 6.4. (B) Time course of the reaction. (C) Reaction after 1 h. Resonances are color-coded to match the peaks associated with the structures in Figure 1A.

Figure 3: Polarimetric profiles of 5.5 mM d-galactaro-δ-lactone without enzyme (black diamonds), with A9CEQ7 (red squares), and with Gci (blue circles). Profile of 5.5 mM d-galactaro-γ-lactone with Gci (green triangles).

Figure 4: Structure of A9CEQ7. (A) Distorted (β/α)8-TIM barrel. A9CEQ7 (cyan) with supposed catalytic resides (yellow). LigI (4d8L) is colored magenta. (B) Superimposition of catalytic residues with those of LigI and the δ-lactone with PDC. Note an asparagine (N240) has replaced an aspartate involved in activating a water at the end of β-strand 8.

Figure 5: Proposed mechanism for A9CEQ7.

Reprinted with permission from Biochemistry. Copyright 2014 American Chemical Society.