title: Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58.
abstract: The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry 46, 9564-9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry 48, 11546-11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, and §Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Fiona P
authors.initials: FP
authors.lastname: Groninger-Poe
authors.forename: Jason T
authors.initials: JT
authors.lastname: Bouvier
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Vetting
authors.forename: Chakrapani
authors.initials: C
authors.lastname: Kalyanaraman
authors.forename: Ritesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.forename: John A
authors.initials: JA
authors.lastname: Gerlt
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D016680:Genome, Bacterial; D006603:Hexuronic Acids / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D013237:Stereoisomerism; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006603:Hexuronic Acids; D013400:Sugar Acids; C007819:galacturonic acid; C000090:galactaric acid; D006836:Hydro-Lyases