Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities

• Published in: PloS one 2017-04, Vol.12 (4), p.e0175193-e0175193
• Main Authors: Pardeshi, Peehu, Rao, K Krishnamurthy, Balaji, Petety V
• Format: Article
• Language: English
• Subjects: Acrylamide; Alignment; Amino Acid Sequence; Amino acids; Arabinogalactan; B7 antigen; Bacon; Bacteria; Biochemistry; Bioengineering; Bioinformatics; Biological activity; Biology and Life Sciences; Biosynthesis; Cadmium; Calcium; Calcium chloride; Campylobacter jejuni - chemistry; Campylobacter jejuni - enzymology; Campylobacter jejuni - genetics; Campylobacter jejuni - metabolism; Carbohydrate Epimerases - chemistry; Carbohydrate Epimerases - genetics; Carbohydrate Epimerases - metabolism; Catalysis; Cell surface; Chemicals; Cholesterol; Cloning; Cloning, Molecular; Computational biology; Computer programs; Cytology; Cytoplasm; Dehydrogenase; Deoxyribonucleic acid; DNA; DNA polymerase; E coli; Enzymatic activity; Enzymes; Functional analysis; Fungi; Gene expression; Gene mapping; Genomes; Genomics; Glucose; High resolution; Homology; Humans; Immunoglobulin E; Infections; Inflammation; Integrated circuits; Kinases; Liquid chromatography; Markers; Metabolism; Molecular weight; Mutagenesis, Site-Directed; Mutants; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; Network analysis; Nucleotide sequence; Peptides; Phenols; Physical sciences; Point Mutation; Prokaryotes; Protein Conformation; Proteins; Proteomics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Research and Analysis Methods; Residues; Sequence Alignment; Site-directed mutagenesis; Spectroscopy; Substrates; Surgical implants; Transcription; Tuberculosis; Tuberculosis - microbiology; UDPglucose 4-Epimerase - chemistry; UDPglucose 4-Epimerase - genetics; UDPglucose 4-Epimerase - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0175193

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.