Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans

Authors: Wagschal, Kurt; Jordan, Douglas; Lee, Charles; Younger, Aunna; Braker, Jay; Chan, Victor

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2014
Publication Date: 12/30/2014
Publication URL: http://dx.doi.org/10.1016/j.enzmictec.2014.12.008
Citation: Wagschal, K.C., Jordan, D.B., Lee, C.C., Younger, A.R., Braker, J.D., Chan, V.J. 2014. Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans. Enzyme and Microbial Technology. 69:62-68.

Interpretive Summary: Pectins from citrus waste processing and sugar beet refining represent a significant untapped biomass resource since the annual U.S. citrus production is ~14 M metric tons/year, and the sugar beet production in the U.S. is ~30 M metric tons/year. Pectins are currently used primarily as animal feed or discarded; the use as livestock feed involves drying the pulp to preclude spoilage, with an attendant energy requirement 30-40% of the total energy input for beet processing, while disposal incurs added expense, and it is thus becoming economically and environmentally desirable to develop value-added products from these bio-resources. 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, which are DOE top value biochemicals from biomass due to potential use in synthetic polyhydroxypolyamides (nylons), and as chelators in e.g. the detergent surfactant market. 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 ºC, and the enzyme from Chromohalobacter was moderately thermostable with Kt(0.5) = 62.2 ºC.

Technical Abstract: 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, which are DOE top value biochemicals 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 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 ºC, and the enzyme from Chromohalobacter was moderately thermostable with Kt(0.5) = 62.2 ºC. 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.