FGD I is an F-420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGDI using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smeginatis was used, soluble protein yields of 7 mg/L of culture were achieved. Both native and selenomethionine-substituted FGDI were obtained by culturing M. smegmatis in auto-induction media protocols originally developed for E coli. Using these media afforded the advantages of decreased handling, as cultures did not require monitoring of optical density and induction, and reduced cost by removing the need for expensive ADC enrichment normally used in mycobacterial cultures. Selenomethionine was efficiently incorporated at levels required for multiwavelength anomalous diffraction experiments used in crystal structure determination. As far as we are aware this is the first protocol for preparation of selenomethionine-substituted protein in mycobacteria. Native and selenomethionine-labeled FGD1 were successfully crystallized by vapor diffusion, with the crystals diffracting to 2.1 angstrom resolution. (c) 2007 Elsevier Inc. All rights reserved.