title: Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus.
abstract: D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5)  = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.
authors.affiliation: USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA, 94710, USA. kurt.wagschal@ars.usda.gov.
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
authors.affiliation: USDA-ARS-NCAUR, 1815 N University Street, Peoria, IL, 61604, USA.
authors.forename: J
authors.initials: J
authors.lastname: Rose Stoller
authors.affiliation: USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Chan
authors.affiliation: USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA, 94710, USA.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Keck Biophysics Facility and Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60201, USA.
authors.forename: Arabela A
authors.initials: AA
authors.lastname: Grigorescu
authors.affiliation: USDA-ARS-NCAUR, 1815 N University Street, Peoria, IL, 61604, USA. douglas.jordan@ars.usda.gov.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015870:Gene Expression; D005813:Genes, Synthetic; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization
chemical_list: D001426:Bacterial Proteins; D011096:Polygalacturonase
keywords: GH28; Galacturonic acid; Hyperthermostable; Pectin; Polygalacturonase; Product inhibition; Substrate inhibition