Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase fromChromohalobacter salexigens

Abstract

Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD + rather than NADP + as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M TrisHCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å . The GluUADH crystal belonged to space group P6 3 , with unit-cell parameters a = b = 122.58, c = 150.49 Å , = 120. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V M ) is 2.78 Å 3 Da À1 . The structure was solved by the single anomalous dispersion method and structure refinement is in progress.

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

“…Induction, discovery, purification and characterization of UDH enzyme of unknown amino acid sequence from Pseudomonas syringae was first reported in 1959 and intermittently thereafter [8,15,16], while recombinant UDH from this organism, identical to that reported on here, has previously been partially characterized [17]. X-ray crystal structures have been solved for UDH from both Chromohalobacter salixigens (pdb code 3AY3) [1,18] and Agrobacterium tumefaciens (pdb code 3RFT) [19]. Reported applications of UDH include development of spectrophotometric single-and coupled-enzyme assays for the quantitation of uronate glycosides and uronic acids [20,21], metabolic engineering of the fungi Hypocrea jecorina and Aspergillus niger [22] and Saccharomyces cerevisiae [23] for conversion of GalUA to galactaric acid, and creation of a synthetic metabolic pathway not observed in nature for the (in vivo) conversion of glucose to glucaric acid in E. coli [24].…”

Characterization of uronate dehydrogenases catalysing the initial step in an oxidative pathway

“…) was performed. Complemented by the Udh of C. salexigens for which a crystal structure is available, a final selection was conducted (Ahn et al ., ). The putative uronate dehydrogenase of F. pelagi HTCC2506 was chosen because of the close proximity to A. tumefaciens C58, which exhibits the highest catalytic activity (Yoon et al ., ).…”

“…). They are all belonging to the short‐chain dehydrogenase/reductase (SDR) family (Ahn et al ., ; Parkkinen et al ., ) and, based on the conserved motifs identified by Jörnvall and Persson (in Persson et al ., ), are part of ‘Intermediate’ family.…”

“…In addition, fungal strains, which are capable to produce meso ‐galactarate by use of uronate dehydrogenase, were developed (Mojzita et al ., ). Recently, the crystal structures of two uronate dehydrogenases from A. tumefaciens and Chromohalobacter salexigens DSM 3043 were elucidated (Ahn et al ., ; Parkkinen et al ., ). The increased interest on enzymes and pathways converting sugar and sugar derivatives reflects the need of biotechnological conversion routes of biogenic raw materials into chemicals and fuels (Grohmann et al ., ; Carvalheiro et al ., ).…”

Biochemical and Structural Characterization of a Uronic Acid Oxidase from Citrus sinensis

“…Yet, for the enzymatic selective oxidation of uronic acids, only a limited number of enzymes has been described. Some bacteria have been shown to produce oxidative enzymes acting on uronic acids, [6,7] employing NAD(P) + as oxidizing cofactor. While dehydrogenases have inherent drawbacks when used as isolated enzymes, several attempts have been made to produce galactaric acid employing whole cells that contain such dehydrogenases [8,9] .…”