title: Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.
abstract: Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: M Ahsanul
authors.initials: MA
authors.lastname: Islam
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Anatoli
authors.initials: A
authors.lastname: Tchigvintsev
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Veronica
authors.initials: V
authors.lastname: Yim
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Mahadevan
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Edwards
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D041862:Chloroflexi / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003198:Computer Simulation; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D007521:Isocitrate Dehydrogenase; D008359:Mannose-6-Phosphate Isomerase