Identification of a Rubredoxin-like Protein Required for Biotin Synthesis in Mycobacteria

Abstract

In Mycobacterium tuberculosis (TB), biotin is crucial for synthesizing glucose, fatty acids, and mycolic acid components of the bacterial cell wall essential for survival and pathogenesis. TB grows in a biotin-deficient environment during infection and must synthesize its own biotin de novo. Biotin synthase catalyzes the final step in biotin synthesis, in which S-adenosylmethionine (SAM) is used to oxidize C-H bonds and a sulfur atom from an iron-sulfur cluster is inserted between two carbon atoms in the biotin precursor, dethiobiotin. In E. coli, biotin synthase also requires the exogenous electron donor flavodoxin, but this protein will not support the activity of the TB enzyme. In mycobacterial genomes, the biotin synthase gene (BioB or Rv1589 in TB) is found adjacent to two uncharacterized genes that code for small proteins with unknown functions (Rv1590 and Rv1591 in TB). Rv1590 codes for a metal-binding protein homologous to rubredoxin domains, suggesting Rv1590 could be involved in electron transfer reactions. This research project focuses on cloning the Rv1590 gene, heterologous expression in E. coli, purification of the resulting metalloprotein, and investigation of the secondary structure, thermal stability, metal content, and redox activity of the purified protein.