1. Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities
- Author
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K. Krishnamurthy Rao, Peehu Pardeshi, and Petety V. Balaji
- Subjects
Protein Structure Comparison ,Glycerol ,0301 basic medicine ,Protein Conformation ,Mutant ,lcsh:Medicine ,Nucleotide sugar ,Biochemistry ,Database and Informatics Methods ,UDPglucose 4-Epimerase ,chemistry.chemical_compound ,Macromolecular Structure Analysis ,Cloning, Molecular ,lcsh:Science ,Peptide sequence ,Liquid Chromatography ,chemistry.chemical_classification ,Multidisciplinary ,Biochemical-Characterization ,Chromatographic Techniques ,Genomics ,Galactose 4'-Epimerase ,Recombinant Proteins ,Cell-Wall ,Actinobacteria ,Physical sciences ,Chemistry ,Mycobacterium-Tuberculosis ,Sequence Analysis ,Research Article ,Protein Structure ,Bioinformatics ,030106 microbiology ,Substrate-Specificity ,Pseudomonas-Aeruginosa ,Monomers (Chemistry) ,Biology ,Research and Analysis Methods ,Campylobacter jejuni ,Escherichia-Coli O86-B7 ,03 medical and health sciences ,Sequence Motif Analysis ,Catalytic triad ,Humans ,Point Mutation ,Tuberculosis ,Polymer chemistry ,Amino Acid Sequence ,Molecular Biology ,Bacteria ,Hexose 4-Epimerases ,lcsh:R ,Organisms ,Campylobacter-Jejuni ,Biology and Life Sciences ,Proteins ,Mycobacterium tuberculosis ,biology.organism_classification ,High Performance Liquid Chromatography ,Enzyme assay ,carbohydrates (lipids) ,030104 developmental biology ,Enzyme ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Glcnac/Glc 4-Epimerase ,lcsh:Q ,NAD+ kinase ,Carbohydrate Epimerases ,Sequence Alignment - Abstract
A bioinformatics study revealed that Mycobacterium tuberculosis H37Rv (Mtb) contains sequence homologs of Campylobacter jejuni protein glycosylation enzymes. The ORF Rv3634c from Mtb was identified as a sequence homolog of C. jejuni UDP-Gal/GalNAc 4-epimerase. This study reports the cloning of Rv3634c and its expression as an N-terminal His-tagged protein. The recombinant protein was shown to have UDP-Gal/Glc 4-epimerase activity by GOD-POD assay and by reverse phase HPLC. This enzyme was shown to have UDP-GalNAc 4-epimerase activity also. Residues Ser121, Tyr146 and Lys150 were shown by site-directed mutagenesis to be important for enzyme activity. Mutation of Ser121 and Tyr146 to Ala and Phe, respectively, led to complete loss of activity whereas mutation of Lys150 to Arg led to partial loss of activity. There were no gross changes in the secondary structures of any of these three mutants. These results suggest that Ser121 and Tyr146 are essential for epimerase activity of Rv3634c. UDP-Gal/Glc 4-epimerases from other organisms also have a catalytic triad consisting of Ser, Tyr and Lys. The triad carries out proton transfer from nucleotide sugar to NAD(+) and back, thus effecting the epimerization of the substrate. Addition of NAD(+) to Lys150 significantly abrogates the loss of activity, suggesting that, as in other epimerases, NAD(+) is associated with Rv3634c.
- Published
- 2017