Role of fatty acyl-CoA synthetase FadD5 and ATPase Mkl in Mycobacterium tuberculosis lipid metabolism and transport

Mycobacterium tuberculosis (Mtb) is a pathogen that causes the infectious disease tuberculosis and is the leading cause of death worldwide. The cell wall of Mtb is made of a protective layer of various lipids and it has adapted itself to survive by metabolizing lipids as a carbon source. Mtb has the ability to overturn the host immune system and trigger granuloma formation where it survives and replicates utilizing lipids from itself and the host. In Mtb there are four mammalian cell entry (mce) operons and each of the mce operons codes for two permease subunits, YrbEA and YrbEB and six substrate-binding proteins MceA-F. All four systems are proposed to be energized by an ATPase, Mkl (also known as MceG). Mkl and YrbE1AB, the ATPase and permease subunits, respectively, form the ABC transporter part of the Mce1 complex which is shown to transport fatty acids across the cell wall of Mtb. FadD5, a putative fatty acyl-CoA synthetase is thought to activate the fatty acids imported by the Mce1 complex for further processing via β-oxidation. We have successfully produced FadD5 in E. coli and purified it. Biophysical characterization shows that the purified FadD5 is well-folded and dimeric in solution. Further, the structures of FadD5 and its variants were determined by X-ray crystallography and small angle X-ray scattering which show that FadD5 is functional as a homodimer and the C-terminal domain is critical for the overall function of the enzyme. Interestingly, the C-terminal domain of FadD5 is cleaved off during crystallization. Biochemical and enzyme kinetic studies show that FadD5 efficiently conjugates CoA to fatty acids of chain length 12 or more. The recombinant expression and purification of a stable Mkl-YrbE1AB complex was challenging. However, a deletion strain of Mtb by deleting the gene mkl (mkl) was generated. A comparative transcriptomic analysis of wild type and mkl strains grown under different lipids (cholesterol and mycolic acid) as the sole carbon source was performed using RNA-sequencing. These studies have provided insight into the changes in transcriptomic level especially due to mycolic acid such as creating a dormancy related phenotype and the importance of Mkl in Mtb.