title: KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.
abstract: Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Joanne K
authors.initials: JK
authors.lastname: Hobbs
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Seunghyae M
authors.initials: SM
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Melissa
authors.initials: M
authors.lastname: Robb
authors.affiliation: Department of Chemistry, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Fraser
authors.initials: F
authors.lastname: Hof
authors.affiliation: Department of Chemistry, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Christopher
authors.initials: C
authors.lastname: Barr
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada;
authors.forename: Kento T
authors.initials: KT
authors.lastname: Abe
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada; MARUM MPG Bridge Group Marine Glycobiology, Center for Marine Environmental Sciences, University of Bremen, 28334 Bremen, Germany; Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany;
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Hehemann
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: McLean
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada.
authors.forename: D Wade
authors.initials: DW
authors.lastname: Abbott
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 3P6, Canada; boraston@uvic.ca.
authors.forename: Alisdair B
authors.initials: AB
authors.lastname: Boraston
mesh_terms: D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005942:Gluconates / Q000378:metabolism*; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D010368:Pectins / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation; D014574:Uronic Acids / Q000378:metabolism*; D015008:Yersinia enterocolitica / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D005942:Gluconates; D006603:Hexuronic Acids; D011134:Polysaccharides; D014574:Uronic Acids; C002957:2-keto-3-deoxygluconate; D010368:Pectins; D020723:Glucuronic Acid
keywords: alginate; pectin; ring opening; tautomerization; uronate