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Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) belongs to the short-chain dehydrogenase superfamily, specifically those acting on the CH-OH group of donor with NAD + or NADP + as acceptor. It is apparently required for the production of D-glucaric acid. AtuUdh-catalyzed reaction is reversible with dual substrate-specific activity (D-galacturonic acid and D-glucuronic acid) in nature. In our study, 34 mutants were pre-screened from 155 mutants generated from AtuUdh (wild-type) and selected 10 structurally stable mutants with increased substrate selectivity. The specificity, efficiency, and selectivity of these mutants for different substrates and cofactors were predicted from 121 docked models using a substrate-imprinted docking approach. Q14F, S36L, and S75T mutants have shown a high binding affinity to D-glucuronic acid and its substrate intermediates such as D-glucaro-1,4-lactone and D-glucaro-1,5-lactone. These mutants exhibited a low binding affinity to the substrate and cofactor required for D-galactaric acid. D34S, N112E and S165E mutants found to show a high selectivity of D-galacturonic acid and its substrate intermediates for D-galactaric acid production. Ser75, Ser165, and Arg174 are active residues playing an imperative role in the substrate selectivity and also contributed in the conjecture the mechanism of transition state stabilization catalyzed by AtuUdh mutants. The present approach was used to reveal the substrate binding mechanism of AtuUdh mutants for a better understanding of the structural basis for selectivity and function., (Copyright © 2019. Published by Elsevier B.V.)

Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide variety of bacterial sources that convert galacturonic acid, the predominate building block of pectin from these plant sources, and glucuronic acid to their corresponding dicarboxylic acids galactarate and glucarate, the latter being a DOE top value biochemical from biomass. The enzymes from Pseudomonas syringae and Polaromonas naphthalenivorans were found to have the highest reported kcat(glucuronic acid) values, on the order of 220-270 s(-1). The thermal stability of this enzyme type is described for the first time here, where it was found that the Kt((0.5)) value range was >20 °C, and the enzyme from Chromohalobacter was moderately thermostable with Kt((0.5))=62.2 °C. The binding mechanism for these bi-substrate enzymes was also investigated in initial rate experiments, where a predominately steady-state ordered binding pattern was indicated., (Published by Elsevier Inc.)