Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
myo-Inositol dehydrogenase and scyllo-inositol dehydrogenase from Lactobacillus casei BL23 bind their substrates in very different orientations
Section snippets
Bacterial strains, plasmids, culture media and chemicals
Lactobacillus casei BL23 was purchased from the Spanish Type Culture Collection (CECT # 5275). IDH-related genes were cloned into the overexpression plasmid pQE-80 L (QIAGEN Inc.) and expressed in Escherichia coli BL21 gold (DE3) competent cells (Novagen). MRS broth (Sigma-Aldrich) was used for culturing L. casei BL23 strains. Materials for LB media and terrific broth (TB) media such as yeast extract, peptone, tryptone and agar were purchased from Becton, Dickinson and Company, (BD) USA and/or
Activity of recombinant L. casei BL23 enzymes
The enzymes encoded by iolG1 and iolG2 from L. casei BL23, which we designated as LcIDH1 and LcIDH2, were each expressed bearing an N-terminal hexahistidine tag in E. coli using the pQE-80L vector. Both enzymes were soluble and showed significant myo-inositol dehydrogenase activity in the presence of NAD, and no activity in the presence of NADP. Initial experiments suggested that the activity was notably lower compared to BsIDH [3], therefore dehydrogenase activity of these enzymes with other
Conclusions
We previously showed that from examining sequences of putative IDH proteins and the associated structures found in the PDB, one could divide these proteins into four subgroups. Whether these subgroups are functionally distinct was and is unknown in the absence of functional information. By this classification system, L. casei IolG1 is a member of subgroup 1, as is BsIDH. L. casei IolG2 is a member of subgroup 2. It is tempting to suggest that this connects structural variation directly to
Note Added in Proof
It has come to the authors attention that the structure of a similar scyllo-inositol dehydrogenase has recently been reported: Fukano K, Ozawa K, Kokubu M, Shimizu T, Ito S, Sasaki Y, et al. (2018) Structural basis of L-glucose oxidation by scyllo-inositol dehydrogenase: Implications for a novel enzyme subfamily classification. PLoS ONE 13(5): e0198010. https://doi.org/10.1371/journal.pone.0198010.
Acknowledgements
We thank the lab of Dr. Janet Hill (Department of Veterinary Medicine, University of Saskatchewan) for providing the genomic DNA from Lactobacillus casei BL23. This project is supported by NSERC grants to Dr. Sanders and Dr. Palmer. We thank the staff of the Canadian Macromolecular Crystallography Facility (CMCF) at the Canadian Light Source for their technical assistance in data collection and processing. The Canadian Light Source is supported by the Canada Foundation for Innovation, the
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These authors contributed equally to this work.