Compounding evidence supports the important role in pathogenesis that the metabolism

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by (genome. of bacteria in the lungs of mice infected with the mutant declined after induction of the cellular immune response demonstrating a persistence phenotype identical to that seen upon mutation of the Mce4 transporter (4). Thiolases catalyze the reverse Claisen condensation step in lipid -oxidation. Similarly, the operon, which encodes L-Asparagine monohydrate six proteins, is important for L-Asparagine monohydrate growth on cholesterol and for mycobacterial survival and encodes homologs of lipid-modifying enzymes (7, 8). The operon (cultures grown in the presence of cholesterol (Scheme 1) (9). Rv3544c and Rv3543c, annotated as potential acyl-CoA dehydrogenases, are encoded by two adjacent cistronic genes. Heterologously co-expressed N-His6 tagged Rv3544c and tagless Rv3543c co-purify upon immobilized metal affinity chromatography, indicating they form a quaternary complex. We demonstrated that the complex is a functional acyl-CoA dehydrogenase that dehydrogenates steroid-derived substrates analogous to the metabolite isolated from an knockout strain (9). Scheme 1 An disrupted strain of accumulates methyl 1-(2-propanoate)-3a-H-4-(3-propanoic acid)-7a-methylhexahydro- 5-indanone when cultured with cholesterol. To our knowledge this was the first report of a heteromeric acyl-CoA dehydrogenase (ACAD). All ACADs characterized to date form homotetrameric assemblies comprised of four ~43 kDa monomers, with one exception. The ACAD sub-families specific for very long chain fatty acids (VLCAD and ACAD9) form homodimers from two Rabbit Polyclonal to IPPK ~73 kDa monomers (10, 11). Herein, we present full enzymatic and biophysical characterization of Rv3543c-Rv3544c and demonstrate that Rv3543c-Rv3544c is an 22 heterotetramer. Neither Rv3543c nor Rv3544c is functionally competent by itself. Extensive sequence alignments of Rv3543c and Rv3544c with acyl-CoA dehydrogenases of known three-dimensional structure reveal that a canonical active site glutamate required for general base catalysis is conserved in Rv3543c, but not in Rv3544c. Mutagenesis in combination with kinetic assays confirmed that Glu241 of Rv3543c is the base required for catalysis by this enzyme. Furthermore, Rv3543c-Rv3544c has only two conserved FAD binding sites per tetramer. Formation of the Rv3543c-Rv3544c complex is required to constitute functional binding sites and consequently catalytic activity. We propose that this unique heteromeric structure is required for effecting dehydrogenation of steroyl-CoA substrates, and may be a motif L-Asparagine monohydrate that is utilized for polycyclic-CoA L-Asparagine monohydrate esters in general. Therefore, we now refer to the Rv3544c and Rv3543c acyl-CoA dehydrogenase genes as and to distinguish them from members of the bacterial acyl Co-A dehydrogenase, gene family. Materials and Methods Materials, strains, media, and general methods Ferricenium hexafluorophosphate and ergocalciferol were purchased from Sigma-Aldrich (St. Louis, MO). Coenzyme A was purchased from MP Biomedicals (Solon, Ohio). Isopropyl -D-1thiogalactopyranoside was from Denville Scientific (Metuchen, NJ). Tryptone, HEPES, TRIS, and ampicillin were purchased from Fisher Scientific (Pittsburgh, PA). L-arabinose, chloramphenicol, and sodium chlorite were purchased from Acros Organics (New Jersey). Tetracycline is from US Biochemical Corp (Cleveland, OH) and kanamycin is from IBI Scientific (Peosta, IA). Yeast extract was purchased from Research Products International Co. (Mount Prospect, IL). iProof DNA polymerase was from Bio-Rad (Hercules, CA). Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and protein ladder were from New England Biolabs (Beverly, MA). HisTrap FF columns, MonoQ, and Superdex 75 HiLoad 16/60 and 10/300 GL columns were from GE Healthcare Biosciences Corp. (Piscataway, NJ). Oligonucleotides were from IDT Inc. (Coralville, IA). Total genomic DNA of H37Rv was obtained from the TB Research Materials Facility at Colorado State University (Fort Collins, CO) (NIAD NO1-AI40091). MALDI mass spectra were acquired on a Bruker Autoflex II TOF/TOF. Big Dye DNA sequencing L-Asparagine monohydrate (Applied Biosystems, Foster City, CA; performed by the Stony Brook University Sequencing Facility) was used to verify the coding sequence of the expression plasmids. BL21(DE3) was obtained from BioRad and chaperone plasmid pG-KJE8 was obtained from Takara..

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