title: Purification, crystallization and structural elucidation of D-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation.
abstract: Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is D-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, D-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-L-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6 Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. D-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Vetting
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Bouvier
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Gerlt
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D010368:Pectins / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D059748:Proteolysis
chemical_list: D001426:Bacterial Proteins; D010368:Pectins; D019746:Intramolecular Oxidoreductases; D002118:Calcium
keywords: Agrobacterium tumefaciens; d-galactaro-1,4-lactone cycloisomerase; d-galacturonate; pectin