Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens

Abstract

D-Galacturonic acid is the main component of pectin, a natural polymer that exists in primary cell walls of terrestrial plants. Citrus peel and sugar beet pulp are cheap raw materials, and both contain a large amount of pectin, which is currently exploited mainly as cattle feed. Pectin has the potential to be an important raw material for biotechnical conversions to fuels and chemicals. The microbial pathways of D-galacturonic acid catabolism have recently been described (1). Two different catabolic pathways, the isomerase pathway and the oxidative pathway, have been found in bacteria. The isomerase pathway (in Escherichia coli) converts D-galacturonic acid into pyruvate and D-glyceraldehyde 3-phosphate. The oxidative pathway has been described for Agrobacterium tumefaciens and Pseudomonas syringae (2, 3). In this pathway, D-galacturonic acid is first oxidized into meso-galactaric acid and then converted in the following step to ␣-ketoglutarate.Uronate dehydrogenase (EC 1.1.1.203) is the key enzyme in the oxidative pathway of D-galacturonic acid catabolism in bacteria. The enzyme catalyzes the oxidation of D-galacturonic acid into D-galactaric acid. Uronate dehydrogenases from A. tumefaciens (4) and P. syringae (5) have been purified and characterized, and the corresponding genes have also been identified (6, 7). A. tumefaciens (Rhizobium radiobacter) uronate dehydrogenase (AtUdh) 2 is specific for NAD ϩ as a cofactor but accepts both D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. AtUdh belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins are NAD(P)(H)-dependent enzymes with a wide spectrum of substrate specificities and also different enzyme classes (21). The sequence identities between the members of the SDR family are low, but they share a similar three-dimensional ␣/␤-structure.To date, no structural information on uronate dehydrogenase is available. Here, we present the first three-dimensional structure of uronate dehydrogenase, namely AtUdh. We determined the crystal structures of AtUdh in the apo-form and the ternary complex with NADH and product at 1.9 and 2.1 Å resolutions, respectively. In addition, we performed a site-directed mutagenesis study of the catalytic residue Tyr-136 and determined the NAD ϩ -bound crystal structure of the inactive mutant Y136A. This crystallographic information has enabled us to identify the active site of the enzyme and the molecular basis for cofactor and substrate recognition. We also propose a structure-based mechanism for the oxidation of D-galacturonic acid. This information can be used to improve the properties of the enzyme, especially the substrate specificity and enzymatic activity.

Purification, crystallization and structural elucidation ofD-galactaro-1,4-lactone cycloisomerase fromAgrobacterium tumefaciensinvolved in pectin degradation

“…Together with previous reports (Taberman et al, 2014;Bouvier et al, 2014;Parkkinen et al, 2011), this work completes the structural elucidation of the first four enzymes used by A. tumefaciens to degrade d-galacturonate by providing the structure of the enzyme that catalyses the third step in the pathway. These structures provide the foundation upon which enzyme engineering could be performed to improve the conversion of d-galacturonate, and ultimately pectin, to biofuels and bulk chemicals.…”

Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

“…To find the galacturonic acid transporter, the genome was scanned for genes known to be involved in galacturonic acid catabolism, searching for nearby putative transporter candidates. This search revealed a region on the linear chromosome encoding proteins previously identified as being involved in galacturonic acid catabolism (23)(24)(25)(26)(27)(28)(29). Nearby are three gene loci (Atu3135, Atu3136, and Atu3137) that encode proteins with homologs to the TRAP family transporters.…”

“…In this pathway, D-galacturonate is first oxidized to galactaro-1,5-lactone by uronate dehydrogenase (UDH), encoded by gene locus Atu3143 (20,21,(23)(24)(25), which is then isomerized to galactaro-1,4-lactone by a recently identified isomerase (encoded by gene locus Atu3138) (26). Galactarolactone cycloisomerase (encoded by gene locus Atu3139) (27) catalyzes the ring opening of galactaro-1,4-lactone to form 3-deoxy-2-keto-L-threo-hexarate, which is converted to ␣-ketoglutarate semialdehyde through the function of a keto-deoxy-D-galactarate (KDG) dehydratase (encoded by gene locus Atu3140) (28,29).…”

Microbial Glucuronoyl Esterases: 10 Years after Discovery

“…Benzyl glucuronate, which is the only synthetic substrate commercially available, was recently suggested for both qualitative and quantitative GE assays, including a uronic acid dehydrogenasecoupled assay (36). The drawback of the coupled assay is the fact that uronic acid dehydrogenase accepts only the ␤-anomer of the de-esterified GlcA (37). Fortunately, there is continuous progress in methods to study GEs.…”