Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

Abstract

We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2⌬ cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2⌬ mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2⌬ cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2⌬ cells also restored de novo lipoic acid synthesis to 92 and 40% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.Mycobacterium tuberculosis causes immense human morbidity and mortality worldwide, and it is thought that about 30 million people have died from tuberculosis in the past decade alone (see references 1 and 41 and citations therein). The World Health Organization estimates that one-third of the human population is infected with M. tuberculosis, which kills more adults than any other single infectious agent (43). Although effective drugs against tuberculosis exist, treatment is extended and arduous, and in certain countries 36% of tuberculosis patients are now infected with isoniazid-or rifampinresistant strains (36). Hence, there is a renewed interest and urgency in developing new therapeutics against M. tuberculosis.An attractive target for therapeutics is represented by the essential process of bacterial fatty acid biosynthesis (10, 24). M. tuberculosis contains a type 2 fatty acid synthase system (FASII) that consists of discrete enzymes, but it also has an additional associative FASI system (5) which is comprised of several enzymatic activities within a single multifunctional synthase. The two FAS systems cooperate in the production of mycolic acids, ...

Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II ofMycobacterium tuberculosis

“…Rv0241c behavior is clearly distinct from that of FAS-II Mt enzymes that display a marked specificity for C 12 to C 20 acyl chains carried by an ACP unit (21,25,28,32). Therefore, the Rv0241c protein cannot belong to the FAS-II Mt system, in sharp contrast to an earlier conclusion (9). Consistently, an Rv0241c ortholog was detected in the Corynebacterium genus, which is devoid of FAS-II system.…”

“…Recently, however, it has been shown that Rv0241c gene was able to complement ⌬htd2 (⌬yhr067w) yeast mutant lacking a functional mitochondrial FAS-II dehydratase (9). Gurvitz et al concluded that Rv0241c encodes a bona fide FAS-II-like 3-hydroxyacyl-thioester dehydratase (9).…”

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

“…In addition to this, reduced levels of ACP derivatives (due to the reduced levels of AcpM) as well as the reduced levels of the HadABC complex enables the catalytic role of HtdX in this alternative FAS II route. Specifically the role of the HtdX has been proposed in the dehydration step of the hydroxyacyl-ester to an enoyl intermediate regularly catalyzed by the HadABC complex ( Figure 5) [63]. HtdX is strictly specific for…”

Design, synthesis and characterization of dual inhibitors against new targets FabG4 and HtdX of Mycobacterium tuberculosis

“…Also, HtdX and FabG4 can plausibly function within a shared pathway as they both belong to single operon. 16,18,20 This type of pair arrangement has been observed in other enzymes of FAS family, including FabG1 (Rv1483) and InhA (Rv1484). 6 These reports and facts have made HtdX a potential target for the drug discovery along with FabG4.…”

“…16 The enzyme is hypothesized to be involved in bypass fatty acid synthesis pathway and in drug resistance of Mtb. 8, 10c, 17, 18 There are eleven putative (R)-specific hydratase/dehydratase candidates identified in Mtb, but only HadB (Rv0636) and…”