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Applied and Environmental Microbiology, August 2008, p. 5078-5085, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00655-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C₂₀ Fatty Acids to C₆₀-to-C₉₀ Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

Aner Gurvitz,^1,2* J. Kalervo Hiltunen,² and Alexander J. Kastaniotis²

Section of Physiology of Lipid Metabolism, Institute of Physiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria,¹ Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland²

Received 18 March 2008/ Accepted 9 June 2008

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We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C₂₀ fatty acids to form C₆₀-to-C₉₀ mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1 cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1 cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C₄ to C₈) than was previously thought (>C₁₂). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

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Mycobacterium tuberculosis is the leading cause of mortality due to an infectious agent worldwide. The World Health Organization estimates that approximately 2 billion people have tuberculosis (53). M. tuberculosis is therefore the biggest killer among human pathogens; it is thought that about 1.7 million people die from tuberculosis every year (www.who.int/tb/publications). Moreover, increasing multiple-drug resistance has contributed significantly to the number of incurable cases, and in some countries up to 36% of patients with tuberculosis are infected with strains resistant to isoniazid (INH) or rifampin (42). These two compounds have been used clinically for several decades and are two of only a few first-line antituberculous drugs. The primary target of INH is InhA (3), and this fact alone elevates InhA to the position of one of most medically significant pathogen proteins. Hence, it is important to use new molecular approaches to study InhA in order to identify novel ways of targeting this enzyme, as well as the processes in which it is involved.

InhA participates in fatty acid biosynthesis (3). Unlike the situation in Escherichia coli, the mycobacterial process is comprised of two systems. M. tuberculosis has a prokaryotic dissociative type 2 fatty acid synthase (FASII) system, in which individual reactions are catalyzed by discrete polypeptides, a process that has been characterized extensively in E. coli and plant plastids. Additionally, M. tuberculosis has an associative type 1 fatty acid synthase (FASI) system (5), which includes several enzymatic activities within a multifunctional homohexamer and resembles the cytosolic synthase in eukaryotes (47). The two systems combine to produce mycolic acids, which are very-long-chain (C₅₄ to C₆₃) -branched, β-hydroxylated fatty acids that act with other factors to form the protective layer around the pathogen, thereby adding to its persistence despite lengthy treatment, and are also associated with its virulence (48).

Based on its fully sequenced genome (13), it has been proposed that M. tuberculosis contains the entire complement of FASII components (48). The 2-trans-enoyl-acyl carrier protein (2-trans-enoyl-ACP) reductase of FASII is represented by InhA, which carries out the final step of the fatty acid elongation process. The M. tuberculosis genome harbors genes for four additional proteins, Rv0927c, Rv3485c, Rv3530c, and Rv3559c, that are all similar to InhA (48) and exhibit about 24 to 26% sequence identity to the latter protein (Fig. 1). It has been proposed that in M. tuberculosis, InhA catalyzes the reduction of 2-trans-enoyl-ACPs with a chain length greater than C₁₂, whereas InhA in Mycobacterium smegmatis acts on C₁₆ thioesters (35).

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FIG. 1. Comparison of M. tuberculosis InhA with four proteins most similar to it: Genedoc-based comparison of the deduced amino acid sequences of InhA (Rv1484) and Rv3485c, Rv0927c, Rv3530c, and Rv3559c. Dashes were added to the sequences to obtain the best fit. Arrows indicate the positions of the conserved amino acid residues required for catalysis (38).

Yeast (Saccharomyces cerevisiae), mammals, and other higher eukaryotes have traditionally been considered organisms that are capable of synthesizing fatty acids only through FASI. This view has very recently been completely overhauled, since an additional mitochondrial FASII has been discovered in both yeast and mammals (2, 28, 32, 37, 50, 55, 56). In the first committed step of S. cerevisiae FASII activity, Hfa1p, representing mitochondrial acetyl coenzyme A (acetyl-CoA) carboxylase (29), converts acetyl-CoA to malonyl-CoA. A malonyl-CoA transferase, mitochondrial Mct1p (45), transfers the C₃ moiety to ACP, a mitochondrial protein encoded by ACP1 (46). Chain elongation begins with the condensation of acetyl-ACP and malonyl-ACP by Cem1p (23), which acts as a mitochondrial 3-oxoacyl-ACP synthase. Mitochondrial Oar1p (45), a 3-oxoacyl-ACP reductase, produces the 3-hydroxyacyl-ACP intermediate, which is then dehydrated by mitochondrial Htd2p (32), a 3-hydroxyacyl-thioester dehydratase, to generate the 2-trans-enoyl-ACP species. The last step in each round of elongation is catalyzed by mitochondrial Etr1p representing 2-trans-enoyl-thioester reductase (50).

A yeast mutant that lacks Etr1p contains abnormally small mitochondria, does not assemble respiratory complexes, and is exclusively fermentative (50, 54). This phenotype can be rescued by supplying the mutant with the gene for fungal or human mitochondrial 2-trans-enoyl-ACP reductase (37, 50). In addition, the etr1 mutant phenotype can also be rescued with a mitochondrially targeted E. coli FabI protein (50), representing a structurally unrelated FASII enoyl-ACP reductase (4). Whereas it has been proposed that mitochondrial FASII is involved in de novo production of the C₈ precursor of lipoic acid (20), mycobacterial FASII is thought to be incapable of de novo synthesis (5).

Here, we used S. cerevisiae as a surrogate for hosting M. tuberculosis protein genes (17). To examine whether InhA could physiologically metabolize short-chain enoyl-ACP substrates and to determine whether there are functional InhA homologues in M. tuberculosis, InhA and the four similar proteins were expressed in the yeast etr1 mutant, and transformed mutant cells were compared to cells of an otherwise isogenic strain expressing the corresponding native enzyme in terms of growth on glycerol, lipoic acid production, assembly of cytochrome complexes, respiration, and the presence of 2-trans-enoyl-thioester reductase activity. The implications of our finding that InhA can participate in de novo lipoic acid synthesis in yeast mitochondria for fatty acid biosynthesis in M. tuberculosis are briefly discussed below.

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Yeast strains, plasmids, and oligonucleotides.
Yeast strains, plasmids, and oligonucleotides used are listed in Table 1. E. coli strain DH10B was used for all plasmid amplification and isolation procedures. Transformation of yeast strains was performed using a previously described method (10).

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TABLE 1. S. cerevisiae strains, plasmids, and oligonucleotides used

Plasmid construction.
DNA manipulation and plasmid construction were performed using standard techniques (1). Plasmid pPLM48, representing a YEp352-based multicopy plasmid (27) carrying the nucleotide sequence for a COQ3-inhA fusion encoding a mitochondrially targeted InhA (mit-InhA) driven by the oleic acid-inducible CTA1 promoter, was constructed as follows. PCR was performed with M. tuberculosis H37Rv genomic DNA using Phusion high-fidelity DNA polymerase (Finnzymes Oy, Espoo, Finland) and oligonucleotides Rv1484 MLS-InhA F and Rv1484 InhA R (Table 1), which introduced a 5' NcoI site and a 3' HindIII site, respectively, at an annealing temperature of 55°C. In amplification products preceded by an engineered NcoI site, the first nucleotide following the incorporated ATG triplet corresponding to the native translational start codon was replaced with G, when necessary, to accommodate the NcoI palindrome CCATGG. Electrophoretic resolution of the PCR mixture on a 0.7% (wt/vol) agarose gel in a buffer comprised of 40 mM Tris-acetate and 1 mM EDTA (pH 8.0) revealed a single amplification product of the correct size (approximately 900 bp), which was excised and purified using QIAquick spin columns according to the manufacturer's instructions (Qiagen, Valencia, CA). Following digestion with NcoI and HindIII restriction enzymes, the amplified inhA DNA was ligated behind the CTA1 promoter to a similarly digested pYE352:mitQOR plasmid (pPLM62) (see below), from which the QOR open reading frame (ORF) encoding E. coli quinone reductase was removed, leaving behind the nucleotides for the Coq3p (30) mitochondrial leader sequence (MLS). Nucleotide sequencing of the inhA insert verified that no mutations were introduced during the amplification process and that the COQ3-inhA junction remained intact.

To generate plasmid pPLM62, the E. coli mitQOR ORF was amplified from genomic DNA using oligonucleotides Ec qor ORF 5' NcoI and Ec qor ORF 3' XhoI and an annealing temperature of 55°C. The amplification product was digested with NcoI and XhoI and ligated to an XbaI- and XhoI-digested pYE352:CTA1 plasmid (16), which was stored as pPLM51 in our strain collection, together with an NheI- and NcoI-delineated nucleotide sequence for the yeast Coq3p MLS, in a triple ligation reaction like that described previously for plasmid pYE352:mitfabI (50). The NcoI-Rv3485c-HindIII (945 bp), BspHI-Rv0927c-XhoI (792 bp), BspHI-Rv3530c-HindIII (783 bp), and NcoI-Rv3559c-HindIII (789 bp) DNA fragments were generated by PCR essentially as described above for COQ3-inhA, using genomic DNA as the template and the corresponding oligonucleotides. The PCR products were inserted using blunt-end ligation into a pBluescript KS II(+) plasmid vector (pKS; Stratagene, La Jolla, CA) that was digested with EcoRV, resulting in plasmids pPLM57, pPLM58, pPLM59, and pPLM60. Following nucleotide sequencing, the inserts were transferred to the corresponding expression plasmids pPLM52, pPLM53, pPLM54, and pPLM55, as described above for COQ3-inhA. The COQ3 junction with the four inserts was verified by nucleotide sequencing. Construction of YEp352:YBR026c expressing S. cerevisiae mitochondrial ETR1 from the CTA1 promoter, which we designated YEp352:ETR1 and stored as pPLM50, has been described previously (50). The steps used to generate the pYE352:CTA1 construct for expressing Cta1p have been described elsewhere (16).

For low-level expression of InhA, PCR was performed with pPLM48 template DNA and oligonucleotides COQ3 MLS 5' NheI and CTA1 term 3' EcoRI, which introduced a 5' NheI site and a 3' EcoRI site, respectively, using an annealing temperature of 55°C. A single amplification product that was 1.3 kb, consisting of the inhA ORF (810 bp), the CTA1 terminator (420 bp), and the COQ3 MLS (60 bp), was resolved using agarose gel electrophoresis, excised, and purified. Following ligation with an EcoRV-digested pKS plasmid to generate pPLM238, the insert was released by digesting the recombinant plasmid with NheI and EcoRI, and after electrophoresis and fragment purification, it was ligated to an XbaI- and EcoRI-digested pPLM239 plasmid, a mutant construct that did not express mitochondrially targeted E. coli FabI (see below), from which the sequence for E. coli fabI was removed, resulting in plasmid pPLM240.

Plasmid pPLM239 was constructed as follows. A COQ3_MLSmut-fabI-CTA1_terminator DNA fragment was generated by PCR amplification using primers COQ3 5' XbaI and CTA1 term 3' EcoRI and template DNA from plasmid pYE352:mitfabI (50), and the product was digested with XbaI and EcoRI prior to ligation. The ScETR1 regulatory region (974 nucleotides upstream of the ETR1 translation initiation codon) was amplified from plasmid pTSV30A:MRF1' (see below) with primers MRF1' GENE 5' SacI/HindIII and MRF1'up 3' XbaI, and the PCR product was digested with XbaI and HindIII prior to ligation. The digested PCR products were simultaneously ligated to a HindIII- and EcoRI-digested YCplac22 plasmid (18) in a triple ligation reaction, resulting in plasmid pPLM239. After sequencing of this construct, we noticed that one of the PCR oligonucleotides was faulty since it had introduced a point mutation into COQ3_MLS, resulting in an in-frame stop codon.

Plasmid pTSV30A:MRF1' was constructed by PCR amplification of the ETR1 gene using the primers mentioned above and template DNA from plasmid YCplac22:MRF1', restriction digestion of the PCR product with SacI and BglII, and ligation of the digested PCR product to a SacI- and BamHI-digested pTSV30A plasmid. Nonintegrating episomal plasmid pTSV30A was generated by J. Pringle and M. Longtine, and its salient features have been described previously (32). Plasmid YCplac22:MRF1' (pPLM241) was constructed by PCR amplification of the ScETR1 gene, including the native promoter and terminator sequences, from yeast genomic DNA using primers MRF1' GENE 5' and MRF1' GENE 3'BglII. The product was digested with HindIII and BglII and ligated to YCplac22 (18) digested with HindIII and BamHI.

Media and growth conditions.
Standard yeast (40) and E. coli (43) media were prepared as described previously. S. cerevisiae strains were propagated on solid rich glucose medium YPD, consisting of 1% (wt/vol) yeast extract, 2% (wt/vol) peptone (YP), 2% (wt/vol) D-glucose, and 2% (wt/vol) agar. Episomal plasmids were maintained in transformed strains using solid SD selective media, consisting of 0.67% (wt/vol) yeast nitrogen base without amino acids, 2% (wt/vol) D-glucose, and 2% (wt/vol) agar and supplemented with yeast synthetic drop-out medium without uracil or tryptophan (Sigma-Aldrich Inc., St. Louis, MO), as required. Cultivation of yeast cells in oleic acid medium for enzyme assays was performed as described previously (22).

Miscellaneous.
For reductase activity assays, 50-ml cultures of oleic acid-grown yeast cells were collected by centrifugation and washed twice in cold distilled water, and 300-µl portions of the freshly pelleted cells were collected in 1.5-ml plastic tubes for further processing. Cells were broken with glass beads in 100 µl breakage buffer that consisted of 50 mM KP_i (pH 7.0), 200 mM KCl, and 0.1% (wt/vol) Triton X-100. Protein concentrations were determined by the method of Bradford (7). Enoyl reductase activity was assayed spectrophotometrically at 23°C as described previously (15). The assay mixture consisted of 50 mM KP_i (pH 7.5), 0.1 mg/ml bovine serum albumin, 125 µM NADH for InhA or 125 µM NADPH for Etr1p, and 60 µM 2-trans-decenoyl-CoA [trans-C_10:1(2)] or 2-trans-hexenoyl-CoA [trans-C_6:1(2)], which was synthesized using the mixed anhydride system (19), as the substrate. Respiration competence was assayed by overlaying cells spotted onto solid SD-tryptophan minus medium with 0.1% (wt/vol) 2,3,5-triphenyltetrazolium chloride (TTC) in 0.067 M phosphate-buffered saline and 1.5% (wt/vol) low-melting-temperature agarose (6). Cytochromes were monitored spectrophotometrically by measuring the absorbance between 640 and 480 nm of yeast cells applied as a thick paste onto the glass surface of an otherwise aluminum cold-temperature cuvette that was chilled in liquid nitrogen (34). The lipoic acid content of yeast strains was monitored with a biological assay described previously (24, 26) using lipoic acid-deficient E. coli strain JRG33-lip9.

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S. cerevisiae etr1 cells expressing M. tuberculosis InhA have 2-trans-enoyl-thioester reductase activity.
To examine whether InhA or the four proteins similar to InhA were synthesized by S. cerevisiae as active 2-trans-enoyl-thioester reductases, the corresponding genes were expressed in yeast cells lacking the native mitochondrial 2-trans-enoyl-thioester reductase Etr1p. This was done by fusing the mycobacterial proteins at their N termini with the cleavable Coq3p MLS that has been demonstrated previously to be sufficient for targeting proteins to yeast mitochondria (30) (i.e., Coq3p_MLS-InhA). We included the predicted MLS cleavage site (MITOPROT II) in our construct (12). Upon entry into the mitochondria, the fused MLS is cleaved off, leaving behind the M. tuberculosis protein preceded by eight amino acids remaining from the MLS fusion (50). The first amino acid following the native methionine of the M. tuberculosis protein was changed to accommodate the NcoI restriction site. As controls, isogenic etr1 cells were transformed with plasmids for expressing either native mitochondrial Etr1p (mit-Etr1p) or peroxisomal catalase A (per-Cta1p). All constructs were tethered behind the yeast CTA1 promoter on URA3-marked multicopy plasmids, and yeast transformants were selected and maintained on glucose medium lacking uracil.

BY4741etr1 cells expressing M. tuberculosis InhA (yPLM14) or one of the four similar proteins (yPLM29, yPLM61, yPLM30, or yPLM31) were examined to determine their enoyl-thioester reductase activity by assaying their contents for the presence of this activity using trans-C_10:1(2) as the substrate. In order to obtain high levels of protein expression from the fatty acid-inducible CTA1 promoter, cells were grown on oleic acid medium overnight prior to breakage with glass beads. The results of the enzyme assays showed that soluble protein extracts with ample mit-InhA generated from strain yPLM14 gave rise to NADH-dependent reduction of trans-C_10:1(2) at a rate of 42.1 ± 5.0 nmol/mg protein min^–1 (mean ± standard deviation; n = 3). Extracts obtained from an isogenic etr1 strain (yPLM26) overexpressing native Etr1p from a similar multicopy plasmid yielded NADPH-dependent reduction of trans-C_6:1(2) at a rate of 49.6 ± 3.8 nmol/mg protein min^–1 (n = 3). No specific enoyl-thioester reductase activity was observed in soluble protein extracts from etr1 cells overexpressing per-Cta1p (yPLM25). Hence, heterologous expression of InhA in mutant etr1 cells resulted in soluble protein extracts containing detectable levels of enoyl-thioester reductase activity. When the shorter trans-C_6:1(2)was used as the substrate, the enoyl-thioester reductase activity in soluble protein extracts enriched with mit-InhA was below the detection limit of the assay used. None of the soluble protein extracts obtained from yeast transformed with plasmids for expression of mitochondrially localized versions of the four proteins resembling InhA contained reductase activity when trans-C_10:1(2) was used as the substrate.

M. tuberculosis InhA restores respiratory growth of S. cerevisiae etr1 cells.
To determine whether the regeneration of 2-trans-enoyl-thioester reductase activity in the etr1 mutant cells attributed to InhA could compensate for the missing native activity, BJ1991etr1 cells were transformed with YCplac22-based low-copy-number centromeric plasmids marked with TRP1 (18) that carried the ETR1 or inhA genes tethered to the yeast ETR1 promoter. Transformants were selected and maintained on synthetic tryptophan-deficient glucose medium. Following overnight growth in this type of liquid medium and 10-fold serial dilution, cultures were spotted onto solid selective medium containing 2% (wt/vol) glucose or synthetic complete medium containing 3% (wt/vol) glycerol, and the plates were incubated at 30°C until single colonies were detectable.

The results demonstrated that mutant etr1 cells expressing low levels of InhA (yPLM242) resembled the self-complemented strain (yPLM243) in that they grew on glycerol as a sole carbon source (Fig. 2A), although the cells expressing InhA gave rise to smaller colonies than the cells expressing Etr1p. As anticipated, mutant cells harboring the plasmid vector (yPLM244) were not able to grow or divide on this nonfermentable medium. The control plate consisting of tryptophan-deficient glucose medium verified both plasmid presence and the evenness of the serial dilution applied to the three strains (Fig. 2B). None of the four InhA-like proteins were able to restore the respiratory growth of the mutant when they were produced as mitochondrial species from genes inserted into multicopy vectors (data not shown).

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FIG. 2. Growth of S. cerevisiae etr1 mutants expressing M. tuberculosis InhA. BJ1991etr1 cells expressing near-physiological levels of native mitochondrial enoyl-thioester reductase (ScEtr1p; positive control) or mitochondrially targeted InhA (MtInhA) or cells harboring the YCplac22 plasmid vector were grown in liquid glucose medium deficient in tryptophan that selected for the presence of the plasmid. Following 10-fold serial dilution (triangle), beginning with an A600 of 1.0, cultures were spotted on solid media. (A) Growth of transformants on synthetic complete medium supplemented with 3% (wt/vol) glycerol (SCglycerol). (B) Growth of transformants on selective medium supplemented with 2% (wt/vol) glucose (SD-Trp). The plates were incubated at 30°C until single colonies appeared, and the results were recorded photographically. (C) Respiratory activity of S. cerevisiae etr1 mutants expressing M. tuberculosis InhA. BJ1991etr1 cells expressing the indicated proteins were grown on SD-Trp medium (panel B) for several days. The plate was overlaid with 0.2% (wt/vol) TTC, and following development of the red chromophore, the results were recorded photographically. The strains used were yPLM242, yPLM243, and yPLM244.

InhA expression repairs the mitochondrial electron transfer chain and restores lipoic acid production in the etr1 mutant.
A hallmark in the recovery of etr1 cells from their fermentative phenotype is the assembly of cytochrome complexes. To examine this phenomenon qualitatively, transformed yeast strains were monitored spectrophotometrically for cytochrome assembly using the low-temperature cuvette method (34). The results demonstrated that BY4741etr1 cells expressing per-Cta1p did not assemble cytochrome b, a, and a₃ complexes (Fig. 3), confirming previous observations (54). The signal for these complexes was more pronounced in mutant cells expressing native mit-Etr1p or mit-InhA. To determine whether the spectrogram obtained for mutant cells producing mit-InhA correlated with a regenerated electron transfer chain, TTC was applied to a duplicate glucose plate. The results demonstrated that mutant BY4741etr1 cells producing InhA were able to reduce TTC to the poorly water-soluble red chromophore, whereas the cells enriched with per-Cta1p were not able to do this (not shown). TTC was also applied to BJ1991etr1 cells expressing near-physiological levels of Etr1p or InhA on the tryptophan-deficient glucose medium mentioned above (Fig. 2C). This showed that even low levels of InhA were sufficient to cause the colonies to turn red, albeit not as efficiently as the mutants rescued with the native protein, whereas dense cells harboring the plasmid vector remained white. Hence, the combined results of these two qualitative methods led us to suggest that the ability of mutant etr1 cells to grow on glycerol was due to the restoration of their mitochondrial respiratory functions.

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FIG. 3. Spectrophotometric assay for mitochondrial cytochrome complexes. Cell pastes from the BY4741etr1 strains with overexpression plasmids were collected from colonies grown on solid glucose medium that was incubated for 4 days at room temperature and applied to the glass face of otherwise aluminum low-temperature cuvettes that were immersed in liquid nitrogen before they were placed in a spectrophotometer for analysis. The pastes were scanned at wavelengths between 480 and 640 nm. The numbers on the left indicate units of absorbance (ABS), and the x axis indicates the wavelength. Peaks corresponding to known cytochromes are indicated by arrows. The strains used were yPLM14, yPLM25, and yPLM26.

Finally, to link InhA expression with fatty acid synthesis in yeast mitochondria, lipoic acid production in the three BY4741etr1 overexpression strains was examined. As a point of reference, lipoic acid production in BY4741 or BJ1991 wild-type cells relying on their native FASII system was determined in a separate representative experiment in duplicate, and the values obtained were 254 and 218 ng lipoic acid per g, respectively (21). The results of this experiment (Table 2) showed that expression of the gene for mit-InhA from a multicopy episomal plasmid in the BY4741etr1 mutant resulted in a level of lipoic acid production that was equivalent to about 60% of the level observed for mutant cells expressing Etr1p. Lipoic acid measurements were also obtained using BJ1991etr1 cells expressing near-physiological levels of the genes for native Etr1p or mit-InhA loaded on low-copy-number centromeric plasmids, as well as cells harboring the plasmid vector (Table 2). The results demonstrated that when the inhA gene was tethered behind the ETR1 promoter on a low-copy-number centromeric plasmid, the lipoic acid production in mutant cells was only 14% of the production in mutant cells expressing Etr1p in the same genetic context. The significance of this result is discussed below.

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TABLE 2. Lipoic acid production in yeast etr1 mutants expressing different proteins

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Here, mycobacterial InhA was shown to function in living yeast cells, as monitored by four separate biochemical and physiological examinations. First, InhA could be processed by the fungal protein synthesis machinery into an active enzyme. This, in turn, resulted in etr1 mutant cells giving rise to soluble protein extracts with an enoyl-thioester reductase activity of 42.1 ± 5.0 mmol/mg protein min^–1 (mean ± standard deviation; n = 3) when trans-C_10:1(2) was used as the substrate. Although the specific enoyl-thioester reductase activity of InhA with the trans-C_6:1(2) substrate was below the detection limit of the assay used, InhA was nevertheless sufficiently active to replace native Etr1p for respiratory growth (see below). In light of the previously described data regarding the in vitro substrate specificity of InhA for medium- and long-chain (>C₁₂) fatty enoyl-thioesters (14, 35, 39, 41, 42) and not the short-chain species (C₈) associated with yeast mitochondrial FASII, the present finding of functional complementation based on a much more sensitive assay relying on living cells offers a significant refinement of our knowledge of what physiological substrates InhA is able to accept in vivo.

Second, modest expression of mitochondrially targeted InhA in etr1 mutant yeast cells resulted in cell growth on solid glycerol medium. Since this occurred with complemented mutant cells producing only low levels of both InhA and lipoic acid, at least compared to the self-complemented strain, this experimental design circumvented possible scenarios in which highly excessive amounts of InhA in the cells might be responsible for a suppressive effect that was not physiological. This represents an interesting and meaningful variation of previous demonstrations of an in vivo function for InhA in M. smegmatis and E. coli, in which InhA was examined with reference to increased INH resistance, since the present situation was a heterologous system outside the prokaryotes.

Third, InhA expression restored the mitochondrial electron transfer chain in the etr1 mutant, as judged by the formation of cytochrome complexes and the reduction of TTC. And fourth, the expression of InhA in the etr1 mutant was sufficient to support the production of lipoic acid. Although exhaustive evidence has led other workers to suggest that InhA acts physiologically only on preexisting substrates with a chain length of C₂₀ or more (48), here we showed that InhA could engage in de novo production of C₈ lipoic acid, in which InhA would need to act on the C₄ species 2-trans-butenoyl-thioester encountered by native Etr1p in the same process.

It is interesting that complementation of the yeast phenotype occurred in a biochemical background lacking AcpM (or any bacterial equivalent), which is important for the function of other FASII enzymes, including KasA/KasB (44), FabD (33), and FabH (8, 11). Moreover, InhA has been shown to interact physically with the latter four proteins in a yeast two-hybrid system and coimmunoprecipitation experiments (51, 52). Nevertheless, InhA was physiologically functional in yeast cells lacking AcpM, as well as all four interacting partners. It is not known whether the components of yeast mitochondrial FASII give rise to higher-order complexes. However, the possibility that InhA formed specific functional protein-protein interactions with fungal FASII enzymes can be virtually excluded, since mycobacterial InhA is a tetramer belonging to the short-chain alcohol dehydrogenase/reductase family of proteins (14, 35, 39, 41, 42), whereas dimeric Etr1p belongs to the medium-chain alcohol dehydrogenase/reductase family of enzymes accepting medium-length carbon chains (49). It is also worth noting that while Etr1p depends on NADPH, InhA requires NADH for catalysis.

The issue of whether InhA has functional homologues was also addressed in the present study. The mycobacterial ORFs corresponding to Rv0927c, Rv3485c, Rv3530c, and Rv3559c were processed and analyzed just like inhA was processed and analyzed. Nucleotide sequencing revealed that the four ORFs were placed correctly behind the sequence for Coq3p MLS in the appropriate expression plasmids, but whether the yeast transcription and protein synthesis machineries acted on these four ORFs to produce mitochondrial proteins was not determined. Nevertheless, we were unable to demonstrate 2-trans-enoyl-thioester reductase activity either in biochemical assays or complementation experiments with the yeast etr1 mutant. M. tuberculosis mutants have been described for Rv0927c and Rv3485c (36), although whether they were deficient in synthesizing mycolic acids was not reported.

In conclusion, this is the first demonstration of a physiological function for M. tuberculosis InhA within the framework of eukaryotic de novo fatty acid biosynthesis. It is at least plausible that the ability to metabolize in vivo very-short-chain fatty enoyl-thioesters might be shared by other M. tuberculosis FASII enzymes in addition to InhA and HtdZ (21). This not only would extend the versatility of yeast FASII mutants to examining the physiological function of the remaining described mycobacterial FASII enzymes, but also might lead to the identification of unknown enzymes. This new application is especially timely since FASII is emerging as an appealing new target for novel therapeutics (9, 25), and no new antimycolates have been developed in the past three decades, despite the pressing need for such development.

 
We thank Johanna Mäkinen from the Mycobacterial Reference Laboratory at the National Public Health Institute in Turku, Finland, for providing M. tuberculosis genomic DNA.

This work was supported by grants from the Academy of Finland and the Sigrid Jusélius Foundation to J.K.H. and by grants P19378-B03 and P19399-B03 from the Austrian Science Fund (FWF) to A.G.

 
* Corresponding author. Mailing address: Medizinische Universität Wien, Zentrum für Physiologie und Pathophysiologie, Institut für Physiologie, Schwarzspanierstraβe 17, 1090 Vienna, Austria. Phone: 43 1 4277 62126. Fax: 43 1 4277 62198. E-mail: aner.gurvitz{at}meduniwien.ac.at

Published ahead of print on 13 June 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, NY.
2
2. Autio, K. J., A. J. Kastaniotis, H. Pospiech, I. J. Miinalainen, M. S. Schonauer, C. L. Dieckmann, and J. K. Hiltunen. 2008. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. FASEB J. 22:569-578.[Abstract/Free Full Text]
3
3. Banerjee, A., E. Dubnau, A. Quemard, V. Balasubramanian, K. S. Um, T. Wilson, D. Collins, G. de Lisle, and W. R. Jacobs, Jr. 1994. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263:227-230.[Abstract/Free Full Text]
4
4. Bergler, H., P. Wallner, A. Ebeling, B. Leitinger, S. Fuchsbichler, H. Aschauer, G. Kollenz, G. Hogenauer, and F. Turnowsky. 1994. Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli. J. Biol. Chem. 269:5493-5496.[Abstract/Free Full Text]
5
5. Bloch, K. 1977. Control mechanisms for fatty acid synthesis in Mycobacterium smegmatis. Adv. Enzymol 45:1-84.[Medline]
6
6. Böker-Schmitt, E., S. Francisci, and R. J. Schweyen. 1982. Mutations releasing mitochondrial biogenesis from glucose repression in Saccharomyces cerevisiae. J. Bacteriol. 151:303-310.[Abstract/Free Full Text]
7
7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
8
8. Brown, A. K., S. Sridharan, L. Kremer, S. Lindenberg, L. G. Dover, J. C. Sacchettini, and G. S. Besra. 2005. Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity. J. Biol. Chem. 280:32539-32547.[Abstract/Free Full Text]
9
9. Campbell, J. W., and J. E. Cronan, Jr. 2001. Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu. Rev. Microbiol. 55:305-332.[CrossRef][Medline]
10
10. Chen, D.-C., B.-C. Yang, and T.-T. Kuo. 1992. One-step transformation of yeast in stationary phase. Curr. Genet. 21:83-84.[CrossRef][Medline]
11
11. Choi, K. H., L. Kremer, G. S. Besra, and C. O. Rock. 2000. Identification and substrate specificity of β-ketoacyl (acyl carrier protein) synthase III (mtFabH) from Mycobacterium tuberculosis. J. Biol. Chem. 275:28201-28207.[Abstract/Free Full Text]
12
12. Claros, M. G., and P. Vincens. 1996. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241:779-786.[Medline]
13
13. Cole, S. T., et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544.[CrossRef][Medline]
14
14. Dessen, A., A. Quemard, J. S. Blanchard, W. R. Jacobs, Jr., and J. C. Sacchettini. 1995. Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science 267:1638-1641.[Abstract/Free Full Text]
15
15. Dommes, V., and W.-H. Kunau. 1984. 2,4-Dienoyl coenzyme A reductases from bovine liver and Escherichia coli. Comparison of properties. J. Biol. Chem. 259:1781-1788.[Abstract/Free Full Text]
16
16. Filppula, S. A., R. T. Sormunen, A. Hartig, W.-H. Kunau, and J. K. Hiltunen. 1995. Changing stereochemistry for a metabolic pathway in vivo. Experiments with the peroxisomal β-oxidation in yeast. J. Biol. Chem. 270:27453-27457.[Abstract/Free Full Text]
17
17. Gerum, A. B., J. E. Ulmer, D. P. Jacobus, N. P. Jensen, D. R. Sherman, and C. H. Sibley. 2002. Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase. Antimicrob Agents Chemother. 46:3362-3369.[Abstract/Free Full Text]
18
18. Gietz, R. D., and A. Sugino. 1988. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527-534.[CrossRef][Medline]
19
19. Goldman, P., and P. R. Vagelos. 1961. The specificity of triglyceride synthesis from diglycerides in chicken adipose tissue. J. Biol. Chem. 236:2620-2623.[Free Full Text]
20
20. Gueguen, V., D. Macherel, M. Jaquinod, R. Douce, and J. Bourguignon. 2000. Fatty acid and lipoic acid biosynthesis in higher plant mitochondria. J. Biol. Chem. 275:5016-5025.[Abstract/Free Full Text]
21
21. Gurvitz, A., J. K. Hiltunen, and A. J. Kastaniotis. 2008. Identification of a novel mycobacterial 3-hydroxyacyl-thioester dehydratase HtdZ/Rv0130 by functional complementation in yeast. J. Bacteriol. 190:4088-4090.[Abstract/Free Full Text]
22
22. Gurvitz, A., H. Rottensteiner, S. H. Kilpeläinen, A. Hartig, J. K. Hiltunen, M. Binder, I. W. Dawes, and B. Hamilton. 1997. The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19. J. Biol. Chem. 272:22140-22147.[Abstract/Free Full Text]
23
23. Harington, A., C. J. Herbert, B. Tung, G. S. Getz, and P. P. Slonimski. 1993. Identification of a new nuclear gene (CEM1) encoding a protein homologous to a β-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae. Mol. Microbiol. 9:545-555.[Medline]
24
24. Hayden, M. A., I. Y. Huang, G. Iliopoulos, M. Orozco, and G. W. Ashley. 1993. Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9. Biochemistry 32:3778-3782.[CrossRef][Medline]
25
25. Heath, R. J., Y. T. Yu, M. A. Shapiro, E. Olson, and C. O. Rock. 1998. Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. J. Biol. Chem. 273:30316-30320.[Abstract/Free Full Text]
26
26. Herbert, A. A., and J. R. Guest. 1970. Turbidimetric and polarographic assays for lipoic acid using mutants of Escherichia coli. Methods Enzymol. 18:269-272.[CrossRef]
27
27. Hill, J. E., A. M. Myers, T. J. Koerner, and A. Tzagoloff. 1986. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163-167.[CrossRef][Medline]
28
28. Hiltunen, J. K., F. Okubo, V. A. Kursu, K. J. Autio, and A. J. Kastaniotis. 2005. Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast. Biochem. Soc. Trans. 33:1162-1165.[CrossRef][Medline]
29
29. Hoja, U., S. Marthol, J. Hofmann, S. Stegner, R. Schulz, S. Meier, E. Greiner, and E. Schweizer. 2004. HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae. J. Biol. Chem. 279:21779-21786.[Abstract/Free Full Text]
30
30. Hsu, A. Y., W. W. Poon, J. A. Shepherd, D. C. Myles, and C. F. Clarke. 1996. Complementation of coq3 mutant yeast by mitochondrial targeting of the Escherichia coli UbiG polypeptide: evidence that UbiG catalyzes both O-methylation steps in ubiquinone biosynthesis. Biochemistry 35:9797-9806.[CrossRef][Medline]
31
31. Jones, E. W. 1977. Proteinase mutants of Saccharomyces cerevisiae. Genetics 85:23-33.[Abstract/Free Full Text]
32
32. Kastaniotis, A. J., K. J. Autio, R. T. Sormunen, and J. K. Hiltunen. 2004. Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology. Mol. Microbiol. 53:1407-1421.[CrossRef][Medline]
33
33. Kremer, L., K. M. Nampoothiri, S. Lesjean, L. G. Dover, S. Graham, J. Betts, P. J. Brennan, D. E. Minnikin, C. Locht, and G. S. Besra. 2001. Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II. J. Biol. Chem. 276:27967-27974.[Abstract/Free Full Text]
34
34. Lindenmayer, A., and R. W. Estabrook. 1958. Low-temperature spectral studies on the biosynthesis of cytochromes in baker's yeast. Arch. Biochem. Biophys. 78:66-82.[CrossRef][Medline]
35
35. Marrakchi, H., G. Laneelle, and A. Quemard. 2000. InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FAS-II. Microbiology 146:289-296.[Abstract/Free Full Text]
36
36. McAdam, R. A., S. Quan, D. A. Smith, S. Bardarov, J. C. Betts, F. C. Cook, E. U. Hooker, A. P. Lewis, P. Woollard, M. J. Everett, P. T. Lukey, G. J. Bancroft, W. R. Jacobs, Jr., and K. Duncan. 2002. Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence. Microbiology 148:2975-2986.[Abstract/Free Full Text]
37
37. Miinalainen, I. J., Z. J. Chen, J. M. Torkko, P. L. Pirilä, R. T. Sormunen, U. Bergmann, Y. M. Qin, and J. K. Hiltunen. 2003. Characterization of 2-enoyl thioester reductase from mammals. An ortholog of YBR026p/MRF1'p of the yeast mitochondrial fatty acid synthesis type II. J. Biol. Chem. 278:20154-20161.[Abstract/Free Full Text]
38
38. Parikh, S., D. P. Moynihan, G. Xiao, and P. J. Tonge. 1999. Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis. Biochemistry 38:13623-13634.[CrossRef][Medline]
39
39. Quemard, A., J. C. Sacchettini, A. Dessen, C. Vilcheze, R. Bittman, W. R. Jacobs, Jr., and J. S. Blanchard. 1995. Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis. Biochemistry 34:8235-8241.[CrossRef][Medline]
40
40. Rose, M. D., F. Winston, and P. Heiter. 1990. Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
41
41. Rozwarski, D. A., G. A. Grant, D. H. Barton, W. R. Jacobs, Jr., and J. C. Sacchettini. 1998. Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279:98-102.[Abstract/Free Full Text]
42
42. Rozwarski, D. A., C. Vilcheze, M. Sugantino, R. Bittman, and J. C. Sacchettini. 1999. Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD⁺ and a C₁₆ fatty acyl substrate. J. Biol. Chem. 274:15582-15589.[Abstract/Free Full Text]
43
43. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
44
44. Schaeffer, M. L., G. Agnihotri, C. Volker, H. Kallender, P. J. Brennan, and J. T. Lonsdale. 2001. Purification and biochemical characterization of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthases KasA and KasB. J. Biol. Chem. 276:47029-47037.[Abstract/Free Full Text]
45
45. Schneider, R., B. Brors, F. Burger, S. Camrath, and H. Weiss. 1997. Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae. Curr. Genet. 32:384-388.[CrossRef][Medline]
46
46. Schneider, R., M. Massow, T. Lisowsky, and H. Weiss. 1995. Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein. Curr. Genet. 29:10-17.[CrossRef][Medline]
47
47. Schweizer, E., and J. Hofmann. 2004. Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiol. Mol. Biol. Rev. 68:501-517.[Abstract/Free Full Text]
48
48. Takayama, K., C. Wang, and G. S. Besra. 2005. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18:81-101.[Abstract/Free Full Text]
49
49. Torkko, J. M., K. T. Koivuranta, A. J. Kastaniotis, T. T. Airenne, T. Glumoff, M. Ilves, A. Hartig, A. Gurvitz, and J. K. Hiltunen. 2003. Candida tropicalis expresses two mitochondrial 2-enoyl thioester reductases that are able to form both homodimers and heterodimers. J. Biol. Chem. 278:41213-41220.[Abstract/Free Full Text]
50
50. Torkko, J. M., K. T. Koivuranta, I. J. Miinalainen, A. I. Yagi, W. Schmitz, A. J. Kastaniotis, T. T. Airenne, A. Gurvitz, and J. K. Hiltunen. 2001. Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence. Mol. Cell. Biol. 21:6243-6253.[Abstract/Free Full Text]
51
51. Veyron-Churlet, R., S. Bigot, O. Guerrini, S. Verdoux, W. Malaga, M. Daffe, and D. Zerbib. 2005. The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions. J. Mol. Biol. 353:847-858.[CrossRef][Medline]
52
52. Veyron-Churlet, R., O. Guerrini, L. Mourey, M. Daffe, and D. Zerbib. 2004. Protein-protein interactions within the fatty acid synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability. Mol. Microbiol. 54:1161-1172.[CrossRef][Medline]
53
53. World Health Organisation. 1997. Anti-tuberculosis drug resistance in the world: the WHO/IUATLD global project on anti-tuberculosis drug resistance surveillance. WHO Global Tuberculosis Programme, Geneva, Switzerland.
54
54. Yamazoe, M., K. Shirahige, M. B. Rashid, Y. Kaneko, T. Nakayama, N. Ogasawara, and H. Yoshikawa. 1994. A protein which binds preferentially to single-stranded core sequence of autonomously replicating sequence is essential for respiratory function in mitochondrial of Saccharomyces cerevisiae. J. Biol. Chem. 269:15244-15252.[Abstract/Free Full Text]
55
55. Zhang, L., A. K. Joshi, J. Hofmann, E. Schweizer, and S. Smith. 2005. Cloning, expression, and characterization of the human mitochondrial β-ketoacyl synthase. Complementation of the yeast CEM1 knock-out strain. J. Biol. Chem. 280:12422-12429.[Abstract/Free Full Text]
56
56. Zhang, L., A. K. Joshi, and S. Smith. 2003. Cloning, expression, characterization, and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein. J. Biol. Chem. 278:40067-40074.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, August 2008, p. 5078-5085, Vol. 74, No. 16
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