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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.
Highlights
As one of the smallest free-living organisms, Dehalococcoides mccartyi are important for their ability to detoxify ubiquitous and stable groundwater pollutants such as chlorinated ethenes and benzenes into benign or less toxic compounds (Maymó-Gatell et al, 1997; Adrian et al, 2000; 2007a; He et al, 2003; Löffler et al, 2012)
The fundamental understanding of D. mccartyi metabolism, including the genes and enzymes involved in metabolic processes, has wide-ranging implications for the remediation of toxic compounds and global element cycling (Gribble, 2012)
We report the heterologous expression and biochemical characterization of the aforementioned putative isocitrate dehydrogenase (IDH) (KB1_0495) and phosphoglucose isomerase (PGI)/phosphomannose isomerase (PMI) (KB1_0553) from D. mccartyi in KB-1 (Hug, 2012; Ahsanul Islam et al, 2014)
Summary
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. 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. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families
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