Article

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Journal of bacteriology (Impact Factor: 2.69). 03/2009; 191(5):1565-73. DOI: 10.1128/JB.00586-08
Source: PubMed

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k(cat)) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K(m)) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k(cat) = 1.9 x 10(2) s(-1) on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

Download full-text

Full-text

Available from: Tae Seok Moon, Jul 16, 2014
0 Followers
 · 
82 Views
  • Source
    • "In bacteria the genes of a catabolic pathway are often organised in an operon. In the case of the udh gene however, it does not seem that the genes of the remaining pathway are in the same operon (Yoon et al. 2008). It might be that the genes necessary for growth on mucic acid are regulated in a different way than the udh and are consequently in a different operon. "
    [Show abstract] [Hide abstract]
    ABSTRACT: There are at least three different pathways for the catabolism of D-galacturonate in microorganisms. In the oxidative pathway, which was described in some prokaryotic species, D-galacturonate is first oxidised to meso-galactarate (mucate) by a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase (EC 1.1.1.203). In the following steps of the pathway mucate is converted to 2-keto-glutarate. The enzyme activities of this catabolic pathway have been described while the corresponding gene sequences are still unidentified. The D-galacturonate dehydrogenase was purified from Agrobacterium tumefaciens, and the mass of its tryptic peptides was determined using MALDI-TOF mass spectrometry. This enabled the identification of the corresponding gene udh. It codes for a protein with 267 amino acids having homology to the protein family of NAD(P)-binding Rossmann-fold proteins. The open reading frame was functionally expressed in Saccharomyces cerevisiae. The N-terminally tagged protein was not compromised in its activity and was used after purification for a kinetic characterization. The enzyme was specific for NAD and accepted D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. NMR analysis showed that in water solution the substrate D-galacturonic acid is predominantly in pyranosic form which is converted by the enzyme to 1,4 lactone of galactaric acid. This lactone seems stable under intracellular conditions and does not spontaneously open to the linear meso-galactaric acid.
    Applied Microbiology and Biotechnology 11/2009; 86(3):901-9. DOI:10.1007/s00253-009-2333-9
  • [Show abstract] [Hide abstract]
    ABSTRACT: D-Glucuronate is a key metabolite in the process of detoxification of xenobiotics and in a recently constructed synthetic pathway to produce D-glucaric acid, a "top value-added chemical" from biomass. A simple and specific assay of D-glucuronate would be useful for studying these processes, but existing assays are either time-consuming or nonspecific. Using uronate dehydrogenase cloned from Agrobacterium tumefaciens, we developed an assay for D-glucuronate with a detection limit of 5 microM. This method was shown to be more suitable for a system with many interfering compounds than previous methods and was also applied to assays for myo-inositol oxygenase activity.
    Analytical Biochemistry 06/2009; 392(2):183-5. DOI:10.1016/j.ab.2009.05.032
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: D-galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of D-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi D-galacturonate is catabolized through a reductive pathway with a D-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on D-galacturonate. The genes of the pathway for D-galacturonate catabolism were upregulated in the presence of D-galacturonate in A. niger, even when the gene for D-galacturonate reductase was deleted, indicating that D-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a D-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of D-galacturonate to galactarate was introduced to both strains with disrupted D-galacturonate catabolism. Both strains converted D-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the D-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.
    Applied and Environmental Microbiology 11/2009; 76(1):169-75. DOI:10.1128/AEM.02273-09
Show more