eukaryotes have long been thought to undertake de novo fatty acid biosynthesis exclusively in their cytosol . this acyl carrier protein-(acp- ) dependent process relies on a single associative type 1 fatty acid synthase ( fasi ) , which accommodates all the necessary enzyme activities . however , this traditional view has since been updated following the discovery of an additional mitochondrial fasii in yeast and mammals ( reviewed in [ 1 , 2 ] ) . fasii represents a dissociated system in which each of the enzyme activities occurs within a discrete protein . although all of the fungal  and most of the mammalian  fasii constituents have been identified , the metazoan 3-oxoacyl - acp reductase component has hitherto not been reported . the present work is concerned with identifying the nematode mitochondrial fasii enzyme corresponding to 3-oxoacyl - acp reductase . the c. elegans genome has been fully sequenced , and a cursory glance at the databases ( wormbase , http://www.wormbase.org/ ) reveals that the nematode contains numerous enzymes with amino acid similarities to the mitochondrial 3-oxoacyl - acp reductase oar1p of saccharomyces cerevisiae fasii . in s. cerevisiae , fasii is critical for mitochondrial function and morphology , and a lesion to any one of the genes encoding fasii proteins results in a respiratory growth phenotype , underdeveloped mitochondria lacking cytochromes and , in some cases , loss of mitochondrial dna [ 1 , 2 ] . here , s. cerevisiae was employed as a heterologous system for determining whether any of the candidate nematode proteins might represent a mitochondrial 3-oxoacyl - acp reductase , by monitoring their ability to replace fungal oar1p . s. cerevisiae oar1 cells heterologously expressing an active nematode 3-oxoacyl - thioester reductase were compared to an otherwise isogenic strain relying on an ectopically expressed native reductase for growth on glycerol , respiration , and lipoic acid production . the results in yeast are discussed with reference to the potential for a dysfunctional fasii in extending nematode lifespan . the escherichia coli strain top10 f was used for all plasmid amplifications and isolations . the mutant yeast strain by4741oar1 ( mata   his31 leu20 expression plasmids were introduced into by4741oar1 cells using a published method , and transformants were enriched on solid sd - ura medium , described below . dna work and plasmid assembly the f09e10.3 and d1054.8 genes were isolated from cdna by polymerase chain reaction using phusion high fidelity dna polymerase ( finnzymes oy , espoo , finland ) and the appropriate oligonucleotide pairs ( table 1 ) . subsequent processing of the resultant amplicons , including their electrophoresis , purification , and insertion into an ecorv - digested pbluescript plasmid vector ( stratagene , la jolla , ca ) was performed as described . the pbluescript inserts were isolated following digestion with xbai and xhoi restriction enzymes , and ligated behind the cta1 promoter to pye352:cta1   from which the cta1 open reading frame encoding yeast peroxisomal catalase a was removed using the same enzymes . construction of plasmid yep352:oar1   expressing yeast oar1 behind the cta1 promoter will be described elsewhere . episomal plasmids based on the ura3-marked yep352 plasmid vector   were retained in transformed s. cerevisiae strains by applying selective pressure using solid sd - ura medium consisting of 0.67% ( wt / vol ) yeast nitrogen base without amino acids , 2% ( wt / vol ) d - glucose , 3% ( wt / vol ) agar , and with all necessary supplements added except for uracil ( sigma - aldrich inc , st . solid synthetic complete glycerol medium ( scglycerol ) was prepared essentially as the aforementioned sd - ura medium , with the exceptions that uracil was added and glucose was replaced with 3% ( wt / vol ) glycerol as the sole carbon source . for high levels of protein expression prior to enzyme assays , yeast cells were grown on oleic acid medium as described . cells were broken with glass beads in a buffer consisting of 50 mm kpi ( ph 7.0 ) , 200 mm kcl , and 0.1% ( wt / vol ) triton x-100 . protein concentrations   and reductase activities   were determined as described . the reductase activity assay mixture consisted of 50 mm kpi ( ph 8.0 ) and 50 g / ml bovine serum albumin , 2.0 l purified protein representing the hydratase 2 domain from human mfe2 ( 1/10 dilution of a 3 mg / ml stock ) , 125 m nad or nadp , and 60 m 2-trans - hexenoyl - coa . in this assay , the reverse reaction was monitored , whereby the 2-trans - hexenoyl - coa substrate was first hydrated by the aforementioned hydratase 2 domain of human mfe2 into the 3-hydroxyacyl - coa , followed by the removal of two electrons from c3 by 3-oxoacyl - thioester reductase leaving behind a carbonyl group within the 3-oxoacyl - coa product . to improve the sensitivity of the reductase assay , which was designed to be performed on crude extracts , the coincidental reduction of nad or nadp into the corresponding nad(h ) and nadp(h ) species was monitored spectrophotometrically at 340385 nm , instead of just 340 nm , using a dual wavelength spectrophotometer ( shimadzu corp . , respiration competence was monitored using an overlay consisting of 0.1% ( wt / vol ) 2,3,5-triphenyltetrazolium chloride ( ttc ) in phosphate - buffered saline and 1.5% ( wt / vol ) low - melting temperature agarose . the content of lipoic acid in yeast strains was estimated using a biological assay relying on lipoic acid - deficient bacterial cells described previously [ 18 , 19 ] . the assay determined the growth of these defective bacterial cells in liquid medium to which extracts were added that were generated from control and tester yeast strains , and the growth values were converted to the amount of lipoic acid present in the yeast cells based on a standard curve . each yeast strain was grown in triplicates for 48 h in liquid sd - ura medium selecting for plasmid presence . acid hydrolysis was carried out in 0.5 ml 9 n h2so4 , and e. coli jrg33 ( lipa9 ) cultures were inoculated to an initial od600 of 0.015 in 2 ml of 1      basal growth medium   containing 50 mm sodium succinate and grown for 3648 h. mitoprot ( http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter ) , psort ii ( http://psort.ims.u-tokyo.ac.jp/form2.html ) , and targetp ( http://www.cbs.dtu.dk/services/targetp/ ) were used to calculate the n - terminal region of proteins that could support a mitochondrial targeting sequence [ 21 , 22 ] and multalin ( http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/npsa/npsa_multalin.html ) and genedoc ( http://www.nrbsc.org/gfx/genedoc/index.html ) to generate the sequence comparison   in figure 1 . the wormbase homology group identifier kog1200 , which refers to  mitochondrial / plastidial beta - ketoacyl - acp reductases ( 3-oxoacyl - acp reductases ) , lists y39a1a.11/dhs-11 and f09e1.03/dhs-25 as potentially encoding this activity in the nematode . however , there are approximately 28 additional genes annotated with the dhs designation ( alcohol dehydrogenase / short chain ) . moreover , the c. elegans genome contains a multitude of genes whose products are homologous to yeast mitochondrial oar1p . to determine which of these genes might encode proteins residing in mitochondria , the mitoprot ( mp ) , psort ii ( ps ) , and targetp ( tp ) algorithms were used to analyze the deduced amino acid sequences of the candidate genes , resulting in a prediction that the sequence relating to f09e10.3 ( mp , 0.9119 ; ps , 0.1 ; tp , 0.540 ) had a probability of representing a mitochondrial protein that was equivalent to or higher than that calculated for the known yeast mitochondrial protein oar1p ( 0.9166 , 0.1 , 0.244 ) . in addition to f09e10.3 , d1054.8 ( 0.9232 , 0.439 , 0.505 ) was also selected for investigation as it was previously chronicled as the sole potential fasii - like 3-oxoacyl - acp reductase gene whose rnai inactivation affected worm longevity . the conceptual translation products of both d1054.8 and f09e10.3 contained amino acid residues that appeared to correspond to the catalytic triad previously characterized in bacterial fabg , albeit only within the amino acid sequence encoded by the latter nematode gene was the distance across the triad region fully conserved ( figure 1 ) . to determine whether f09e10.3 or d1054.8 might code for 3-oxoacyl - acp reductases , the genes were expressed behind the promoter of yeast cta1 encoding peroxisomal catalase a ( cta1p ) , which is only moderately derepressed on nonfermentable carbon sources such as glycerol , used here for complementation assays . on glycerol medium , yeast cells shift their metabolism to respiration , which requires an operational fasii within functional mitochondria [ 1 , 2 ] . an extra fungal mitochondrial leader sequence was not fused to the nematode proteins . yeast oar1 mutant cells were transformed with plasmids expressing fungal oar1p ( positive control ) , native cta1p ( negative control ) , or each of the nematode f09e10.3 or d1054.8 protein products and were applied to solid scglycerol medium . this demonstrated that only cells expressing f09e10.3 or oar1 could grow and divide ( see below ) . to assess the efficiency of the nematode f09e10.3 protein at replacing yeast oar1p , the aforementioned transformants were propagated on sd - ura medium , and following tenfold serial dilution , cultures were spotted onto sd - ura or scglycerol media , and the plates were incubated at 30c until single colonies were detectable . the results in figure 2 demonstrated that like the situation with the self - complemented strain , mutant cells expressing f09e10.3 could thrive on glycerol , whereas those mutant cells expressing cta1 were not able to proliferate on this medium type . hence , the respiratory - deficient phenotype of mutant oar1 cells was rescued following the expression of f09e10.3 , indicating functional complementation . yeast cells lacking the native oar1 gene have been shown before to lack assembled cytochrome complexes , and so their rehabilitation to normal respiratory growth was expected to occur contemporaneously with the assembly of cytochrome complexes and the regeneration of the electron transport chain . to examine whether mutant cells expressing oar1p or the f09e10.3 protein contained a regenerated electron transfer chain , respiration competence was assayed by applying to the cells spotted onto solid sd - ura medium an overlay consisting of 2,3,5-triphenyltetrazolium chloride ( ttc ) . the results revealed that only mutant oar1 cells expressing oar1p or the f09e10.3 protein could rapidly metabolize ttc to generate the red chromophore , whereas those expressing cta1p were essentially white ( lower panel ; figure 2 ) . fasii enzyme activities can often be measured in soluble protein extracts made from mutant yeast cells ectopically expressing heterologous proteins . for example , enzyme activities for 2-trans - enoyl - thioester reductase can be detected in protein extracts made from yeast etr1 mutant cells producing s. cerevisiae etr1p , candida tropicalis etr1p or etr2p [ 25 , 26 ] , and mycobacterium tuberculosis inha . although discernible levels of 3-hydroxyacyl - thioester dehydratase activity can be measured in extracts made from yeast htd2 mutants producing mycobacterial hadab , hadbc , htdx , htdy , and htdz [ 27 , 28 ] , the sole documented measurement of dehydratase activity in mutant cells enriched for native htd2p could only be undertaken following the laborious preparation of pure mitochondria . to examine whether the f09e10.3 protein contained reductase activity , yeast cells were grown in triplicate cultures on oleic acid medium   under which conditions the cta1 promoter is highly induced . oleic acid has not been shown previously to interfere with yeast fasii [ 9 , 25 , 27 , 28 ] . the cells were broken with glass beads , and the ensuant soluble protein extracts were subjected to spectrophotometric analysis for reductase activity ( in the reverse orientation ) , which ostensibly could depend on either nad or nadp as co - factor . enzyme assays performed on extracts from oar1 cells over - expressing native oar1p , a presumed nadp - dependent enzyme , did not yield appreciable levels of activity using the present assay . likewise , mutant cells over - expressing cta1p also failed to produce extracts with a specific reductase activity . on the other hand , soluble protein extracts obtained from mutant cells over - expressing the f09e10.3 protein gave rise to an nad - dependent oxidation of the 2-trans - hexenoyl - coa substrate at the rate of 0.97      0.18 ( s.d . nadp did not act as co - factor when examining the nematode protein for reductase activity . hence , ectopic expression of c. elegans f09e10.3 in yeast oar1 mutant cells resulted in clearly discernible levels of catalytic activity in an assay designed to expose 3-oxoacyl - thioester reductases . lastly , to couple the expression of the f09e10.3 protein with fatty acid biosynthesis in yeast mitochondria , lipoic acid synthesis was measured in these strains . the results showed that the negative control strain ( oar1 cells expressing peroxisomal catalase a ) yielded extracts supporting only a minimal growth level of the mutant bacterial cells that was equivalent to 25      3 ng lipoic acid per gram wet weight yeast cells , whereas levels of lipoic acid in mutant cells expressing fungal oar1p or the nematode f09e10.3 protein gave rise to a bacterial growth that corresponded to 138      22 or 80      4 ng lipoic acid per or gram wet weight , respectively . hence , the combined results demonstrated that mutant oar1 cells could grow on glycerol since their formerly defective mitochondrial functions were restored due to the expression of the full - length f09e10.3 protein , indicating that it represented a putative mitochondrial 3-oxoacyl - thioester reductase . the present study demonstrated a physiological function for the gene product of c. elegans f09e10.3/dhs-25 as a possible 3-oxoacyl - thioester reductase of mitochondrial fasii . the f09e10.3 protein could reach its designated subcellular compartment in fungi without resorting to a dedicated yeast mitochondrial targeting sequence , so as to replace the native mitochondrial 3-oxoacyl - acp reductase oar1p in s. cerevisiae oar1 cells . the efficiency of this interchange was underscored by monitoring f09e10.3-complemented yeast mutant cells for respiratory growth on glycerol medium , metabolism of ttc , and synthesis of lipoic acid . although the corresponding fasii protein in protozoans has been exposed previously , for example , plasmodium falciparum   fabg was crystallized and characterized enzymatically   and potential oar1p homologues in trypanosome brucei were shown to be mitochondrial , f09e10.3 represents the first metazoan 3-oxoacyl - thioester reductase gene to be positively identified ( see note added in proof ) . wormbase reports no rnai phenotypes for f09e10.3 , but this could be due to redundancy as the result of additional mitochondrial reductases not yet disclosed . although all previously studied 3-oxoacyl - thioester reductases have been shown to depend on nadp(h ) for catalysis , the finding that the f09e10.3 protein relied on nad(h ) was not altogether surprising . this is because other mitochondrial enzyme activities , such as isocitrate dehydrogenase , are also known to require each of these two cofactors . wormbase lists at least 10 potential nad(h)-dependent and an additional 10 potential nadp(h)-dependent isocitrate dehydrogenases ( gene onthologies go:0004449 and go:0004450 , resp . ) . it is tempting to speculate that future studies will uncover additional 3-oxoacyl - thioester reductases in metazoan mitochondria that would similarly depend on nad(h ) rather than on nadp(h ) . the role of mitochondrial processes in affecting nematode longevity has been studied extensively using rnai technology [ 23 , 33 , 34 ] . dysfunctional mitochondrial activities that have been previously linked to a prolonged nematode lifespan include the electron transport chain , rna editing , and cytochrome complex formation . however , since mitochondrial fasii has hitherto not been identified in nematodes , its involvement in c. elegans lifespan extension could not be specifically addressed . if the lifespan extension observed with the rnai intervention of c. elegans w09h1.5 , which encodes a potential homologue of the aforementioned yeast etr1p , is in any way indicative , then a dysfunctional mitochondrial fasii might be conducive to longer nematode life . worm longevity notwithstanding , the issue is that yeast fasii mutants will likely continue to play an important role in exposing fasii enzymes in metazoan mitochondria . note added in proofidentification of human nad(h)-dependent 3-oxoacyl - acp reductase ,   hskar , of mitochondrial fasii , and the description of pye352:oar1 have been published online . identification of human nad(h)-dependent 3-oxoacyl - acp reductase ,   hskar , of mitochondrial fasii , and the description of pye352:oar1 have been published online .
caenorhabditis elegans f09e10.3 ( dhs-25 ) was identified as encoding a 3-oxoacyl - thioester reductase , potentially of the mitochondrial type 2 fatty acid synthase ( fasii ) system . 
 mitochondrial fasii is a relatively recent discovery in metazoans , and the relevance of this process to animal physiology has not been elucidated . a good animal model to study the role of fasii is the nematode c. elegans . 
 however , the components of nematode mitochondrial fasii have hitherto evaded positive identification . 
 the nematode f09e10.3 protein was ectopically expressed without an additional mitochondrial targeting sequence in saccharomyces cerevisiae mutant cells lacking the homologous mitochondrial fasii enzyme 3-oxoacyl - acp reductase oar1p . 
 these yeast oar1 mutants are unable to respire , grow on nonfermentable carbon sources , or synthesize sufficient levels of lipoic acid . 
 mutant yeast cells producing a full - length mitochondrial f09e10.3 protein contained nad+-dependent 3-oxoacyl - thioester reductase activity and resembled the corresponding mutant overexpressing native oar1p for the above - mentioned phenotype characteristics . 
 this is the first identification of a metazoan 3-oxoacyl - thioester reductase ( see note added in proof ) .