Mycobacterium　tuberculosis　(Mtb)　is　a　crucial　and　highly　destructive　intracellular　pathogen　responsible　for　causing　tuberculosis　(TB).　The　emergence　and　dissemination　of　multi-drug　resistant　Mtb　has　further　aggravated　the　TB　crisis,　leading　to　high　mortality.　Mtb　FadD2　is　a　fatty　acyl-coenzyme　A　(CoA)　synthetase　that　modifies　the　cell　envelope　and　plays　an　important　role　in　reducing　Mtb　susceptibility　to　pyrazinoic　acid　(POA).　However,　the　functional　mechanism　of　Mtb　FadD2　remains　poorly　understood.　Here,　we　successfully　expressed,　purified　and　obtained　monomeric　FadD2　by　using　buffer　(500　mM　NaCl,　20　mM　Tris-HCl,　pH7.4　and　5　%　glycerol).　Palmitate　was　found　to　be　the　optimal　substrate　for　FadD2.　Fatty　acyl-CoA　synthetase　activity　reached　maximum　at　450　μM　palmitate,　and　the　Km　value　was　318.2　μM　for　palmitate.　The　results　of　mutation　experiments　indicated　the　critical　role　of　T370　and　K551　in　the　enzymatic　activity　of　FadD2.　Our　work　provides　a　guideline　and　concept　for　the　development　of　novel　drugs　against　Mtb.　©　2023　Elsevier　Inc.