Studies of Mycobacterium tuberculosis desaturases and their electron transfer partners

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, contains three fatty acid desaturase homologs, named DesA1, DesA2 and DesA3. DesA1 and DesA2 are proposed to catalyze two essential desaturation steps during the biosynthesis of mycolic acids, a major constituent of the durable mycobacterial cell wall. DesA3 has been identified as a stearoyl-CoA Delta9 desaturase, which produces oleic acid, a major constituent of mycobacterial membrane phospholipids and triglycerides. This thesis is focused on the characterization of these three desaturases, and their electron transfer partners.;The first part of this thesis provides a characterization of the DesA3 system. Genome analysis and biochemical assays revealed that the oxidoreductase Rv3230c functions as a biologically relevant and highly specific electron transfer partner for DesA3. GFP reporter assay revealed two promoter sequences for the DesA3 operon and some patterns for their transcriptional regulation. Both DesA3 catalytic activity assays and GFP-based fluorescence studies revealed that a protein degradation process directed at the C-terminus post-translationally regulates DesA3 activity. GFP-based fluorescence studies were also used to determine the amino acid specificity of the protein degradation process. The stabilizing influence of a gene in the 3' position of polycistronic transcript was identified in Mycobacterium smegmatis, and this property was used in the development of a tunable gene co-expression system for this genus. The function of the co-expression system was demonstrated by studies of the in vivo co-expression of Rv3230c and DesA3, leading to tunable changes in the catalytic activity.;In the second part of the thesis, characterizations of the DesA1 and DesA2 systems are presented. An extensive analysis of the M. tuberculosis genome revealed several oxidoreductases and ferredoxins that might serve as electron transfer partners for DesA1 and DesA2. The expression of DesA1, DesA2 and their potential electron transfer partners was accomplished by using a variety of expression systems and methods. In addition, a new method was developed to over-produce holo-AcpM and intermediate chain acyl-AcpM. It is thought these preparations may be used as substrates for further elongation studies by mycobacterial fatty acid synthetase II system, and thus provide elusive long-chain acyl-AcpM for studies DesA1 and DesA2 function.