Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi
journal contributionposted on 07.11.2017, 11:17 by Ahsan IslamAhsan Islam, Anatoli Tchigvintsev, Veronica Yim, Alexei Savchenko, Alexander F. Yakunin, Radhakrishnan Mahadevan, Elizabeth A. Edwards
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.
This research was funded by the University of Toronto, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Government of Canada through Genome Canada and Ontario Genomics Institute (2009-OGI-ABC-1405) and the United States Department of Defense Strategic Environmental Research and Development Program (SERDP). MAI was funded by the Ontario Graduate Scholarship (OGS), the SERDP and Genome Canada funds to EAE and the departmental faculty start-up funds to RM.
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