In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

Abstract

DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A ⌬ 9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His 6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ϳ10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

Global Study of IS 6110 in a Successful Mycobacterium tuberculosis Strain: Clues for Deciphering Its Behavior and for Its Rapid Detection

“…The TbD1 region was absent from the Zaragoza strain, and the mgtC 182 (Arg-His), ogt 44 (Thr-Ser), and ung 501 (Leu-Leu) SNPs characteristic of the Haarlem genotype were not present, which allowed us to classify Zaragoza as a non-Haarlem but "modern" strain of M. tuberculosis.…”

“…Its product is a stearoyl-coenzyme A desaturase (DesA3) implicated in the synthesis of oleic acid and considered to be essential for intracellular growth in macrophages (44,45). In addition, DesA3 is a target of the secondline antituberculosis drug isoxyl, which was used together with isoniazid in multiple-drug therapy in the 1960s (43,44).…”

“…Its product is a stearoyl-coenzyme A desaturase (DesA3) implicated in the synthesis of oleic acid and considered to be essential for intracellular growth in macrophages (44,45). In addition, DesA3 is a target of the secondline antituberculosis drug isoxyl, which was used together with isoniazid in multiple-drug therapy in the 1960s (43,44). Isoxyl inhibits the synthesis of oleic and mycolic acids (43) and was demonstrated to be effective against various clinical isolates of multidrug-resistant strains of M. tuberculosis (46).…”

“…TbD1 deletion results in an amplicon size of 400 bp, whereas the presence of the TbD1 region results in an amplicon size of 2,153 bp. Three single nucleotide polymorphisms (SNPs) described as markers of the M. tuberculosis Haarlem genotype strains, i.e., mgtC 182 (Arg-His) (23), ogt 44 (Thr-Ser), and ung 501 (LeuLeu) (24), were studied by PCR and DNA sequencing using the primers described in Table 1.…”

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

“…Other studies in murine microsomes [11] and differentiated mouse 3L3-L1 adipocytes [15] showed post-translational proteolytic processing of the N-terminus of the enzyme, subsequent inactivation, and proteosome-directed degradation. Interestingly, DesA3, part of the stearoyl-CoA desaturase complex from Mycobacterium tuberculosis [16, 17], is also targeted for rapid proteolytic degradation, but in this case by a prokaryotic degradation complex with specificity for the C-terminus [18]. The multiple layers of regulation of desaturase activity, in diverse organisms, emphasize the importance of this enzyme in cellular function [7], but also complicate recombinant expression using living hosts.…”