title: Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans.
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, 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.
authors: USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA. Electronic address: kurt.wagschal@ars.usda.gov.
authors: Kurt
authors: K
authors: Wagschal
authors: USDA Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
authors: Douglas B
authors: DB
authors: Jordan
authors: USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA.
authors: Charles C
authors: CC
authors: Lee
authors: USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA.
authors: Aunna
authors: A
authors: Younger
authors: USDA Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
authors: Jay D
authors: JD
authors: Braker
authors: USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA.
authors: Victor J
authors: VJ
authors: Chan
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055592:Biophysical Phenomena; D055353:Chromohalobacter / Q000201:enzymology / Q000235:genetics; D042621:Comamonadaceae / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D044203:Pseudomonas mendocina / Q000201:enzymology / Q000235:genetics; D044224:Pseudomonas syringae / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C023280:uronate dehydrogenase; D000445:Aldehyde Oxidoreductases
keywords: Galactaric acid; Galacturonic acid; Glucaric acid; Glucuronic acid; Steady-state ordered binding pattern; Thermal stability; Uronate dehydrogenase