Expression, Purification, and Bioinformatic Prediction of Mycobacterium tuberculosis Rv0439c as a Potential...

Although the genome of Mycobacterium tuberculosis (Mtb) H37Rv, the causative agent of tuberculosis, has been repeatedly annotated and updated, a range of proteins from this human pathogen have unknown functions. Mtb Rv0439c, a member of the short-chain dehydrogenase/reductases superfamily, has yet to be cloned and characterized, and its function remains unclear. In this work, we present for the first time the optimized expression and purification of this enzyme, as well as bioinformatic analysis to unveil its potential coenzyme and substrate. Optimized expression in Escherichia coli yielded soluble Rv0439c, while certain tag fusions resulted in insolubility. Sequence and docking analyses strongly suggested that Rv0439c has a clear preference for NADP⁺, with Arg53 being a key residue that confers coenzyme specificity. Furthermore, functional prediction using CLEAN and DEEPre servers suggested that this protein is a potential NADP⁺-retinol dehydrogenase (EC No. 1.1.1.300) in retinol metabolism, and this was supported by a BLASTp search and docking studies. Collectively, our findings provide a solid basis for future functional characterization and structural studies of Rv0439c, which will contribute to enhanced understanding of Mtb biology.