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Mycothiol S -transferase (MST) (encoded by the rv0443 gene) was previously identified as the enzyme responsible for the transfer of Mycothiol (MSH) to xenobiotic acceptors in Mycobacterium tuberculosis ( M.tb ) during xenobiotic stress. To further characterize the functionality of MST in vitro and the possible roles in vivo , X-ray crystallographic, metal-dependent enzyme kinetics, thermal denaturation studies, and antibiotic MIC determination in rv0433 knockout strain were performed. The binding of MSH and Zn 2+ increases the melting temperature by 12.9 °C as a consequence of the cooperative stabilization of MST by both MSH and metal. The co-crystal structure of MST in complex with MSH and Zn 2+ to 1.45 Å resolution supports the specific utilization of MSH as a substrate as well as affording insights into the structural requirements of MSH binding and the metal-assisted catalytic mechanism of MST. Contrary to the well-defined role of MSH in mycobacterial xenobiotic responses and the ability of MST to bind MSH, cell-based studies with an M.tb rv0443 knockout strain failed to provide evidence for a role of MST in processing of rifampicin or isoniazid. These studies suggest the necessity of a new direction to identify acceptors of the enzyme and better define the biological role of MST in mycobacteria., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)