Inducible 1-Oxidation Pathway in Neurospora crassa

Transcription

1 JOURAL OF BACTERIOLOGY, Jan. 1985, p /85/ $02.00/0 Copyright 1985, Amerian Soiety for Mirobiology Vol. 161, o. 1 Induible 1-Oxidation Pathway in eurospora rassa CHRISTIE KIOKA AD WOLF-H. KUAU* Institut fur Physiologishe Chemie, Abteilung fur aturwissenshaftlihe Medizin, Ruhr-Universitat Bohum, D4630 Bohum 1, Federal Republi of Germany Reeived 23 April 1984/Aepted 13 July 1984 An induible,3-oxidation system was demonstrated in a partiulate fration from eurospora rassa. The ativities of all individual 1-oxidation enzymes were enhaned in ells after a shift from a surose to an aetate medium. The indution was even more pronouned in transfer to a medium ontaining oleate as sole arbon and energy soure. Sine an ayl-oenzyme A (CoA) dehydrogenase was deteted instead of ayl-coa oxidase, the former enzyme seems to atalyze the first step of the "-oxidation sequene in. rassa. After isopyni entrifugation in a linear surose gradient, the intraellular organelles housing the fatty aid degradation pathway osedimented (1.21 glm3) with the glyoxylate bypass enzymes isoitrate lyase and malate synthase and were learly resolved from both mitohondrial marker enzymes (1.19 g/m3) and atalase (1.26 g/m3). On the basis of biohemial as well as morphologial properties, these partiles from. rassa have reently been designated as glyoxysome-like partiles (G. Wanner and T. Theimer, Ann..Y. Aad. Si. 386: , 1982). The failure to detet atalase, urate oxidase, and ayl-coa oxidase indiate that these glyoxysome-like mirobodies in. rassa lak peroxisomal funtion and thus are learly different from the various mirobodies reported so far to ontain a 1-oxidation pathway. In ontrast to the large body of information about the degradation of fatty aids in animal (16, 17, 20) and plant ells (2, 6, 14, 28), muh less is known about this pathway in euaryoti miroorganisms (10, 22, 25). An induible peroxisomal 1-oxidation system has been reported for some yeasts of the Candida genus (10, 25), whereas in the iliate Tetrahymena pyriformis, both mitohondrial and peroxisomal 13-oxidation systems have been found (4). A reent report by Hii and Courtright (18) reported that the filamentous fungus eurospora rassa is able to use longhain fatty aids as the sole arbon and energy soure, suggesting the existene of a 13-oxidation pathway. Their results onerning the induibility of individual enzymes were, however, ambiguous, sine some of them showed no signifiant indution. The intraellular loalization of these enzymes, and thus a distintion between mitohondrial and non-mitohondrial p-oxidation, has not been reported. With respet to intraellular ompartmentation,. rassa appears espeially interesting, beause reently, in addition to mitohondria, two types of mirobody-like organelles have been desribed (27, 30). In the ourse of our studies of the strutural organization of 1-oxidation systems, we have started to investigate the fatty aid degradation pathway in. rassa. In this report we wish to ommuniate: (i) the existene of an induible 13-oxidation system in. rassa, (ii) the exlusive loalization of this pathway in glyoxysome-like mirobodies, and (iii) that this 1-oxidation system does not ontain ayl-oenzyme A (CoA) oxidase as do all other 1-oxidation systems so far deteted in mirobodies (10, 14, 17, 28). MATERIALS AD METHODS Materials. The substanes used in these investigations were obtained from the following soures: AD, flavin adenine dinuleotide, CoA, and 3-hydroxyayl-CoA dehydrogenase, Boehringer Mannheim, Mannheim, Federal Republi of Germany (FRG); bovine serum albumin, Triine, DL-isoitrate, sodium glyoxylate, and Tergitol P-40, Sigma * Corresponding author. Chemie, GmbH, Munih, FRG; iodo-nitrotetrazolium hloride and Triton X-100 (Serva, Heidelberg, FRG; dithioerythritol, Biomol GmbH, Ilvesheim, FRG; phenylhydrazine hydrohloride, Ega-Chemie, Steinheim, FRG; and 8-dimethylamino-2,3-benzophenoxazinium hloride (Meldolablau) was a gift from Sandoz AG, Basel, Switzerland. All other hemials were purhased from E. Merk, Darmstadt, FRG. Unsaturated fatty aids as well as ayl-coa thioesters were prepared as reported previously by Kunau and Dommes (19). Aetyl-CoA was synthesized by the method of Simon and Shemin (24). Growth and ulture onditions.. rassa wild type (strain 740R8-la) was obtained from the Deutshe Sammlung von Mikroorganismen, Gottingen, FRG. Conidia were harvested from stok ultures grown on a potato-surose agar (0.4% infusion from potato, 2% surose, 0.5% yeast extrat, 1.5% agar). Hyphae were grown on Vogels medium (29) supplemented with different arbon soures. After inoulation of 105 onidia per ml, the ultures were shaken at 25 C in a ew Brunswik water bath. For indution experiments as well as for separation of ell organelles, hyphae were first grown on 2% surose for 24 h, harvested by filtration on a Buhner funnel, suspended in a medium ontaining either 40 mm aetate or 1 mm oleate-1% Tergitol P-40, and shaken for another 12 h. For determination of growth urves, hyphae were grown in 2% surose or 1 mm oleate-1% Tergitol by the method of Hii and Courtright (18). Myelia were olleted on a filter paper and dried for 12 h at 80 C. Conidia and hyphae were routinely examined for baterial ontamination. Cell homogenization. Hyphae were harvested by filtration, washed three times with water, and ground with quartz sand (1 g/g wet weight) in isolation medium (4 ml/g wet weight) at 4 C for a. 3 min. The isolation medium onsisted of 150 mm Triine (ph 7.4), 0.44 M surose, 10 mm KCI, 5 mm MgCl2, 153 and 1 mm EDTA. The homogenate was squeezed through four layers of heeseloth and entrifuged for 5 min at 2,500 x g. The resulting supernatant was taken as rude extrat.

2 154 KIOKA AD KUAU.4 n 0.4-_ C) E >11 LL 10 1 I I I Time of growth on oleate [hi FIG. 1. Indution of various enzyme ativities by shifting. rassa ells from a medium ontaining surose (2%) to a medium ontaining 1 mm oleate-1% Tergitol as arbon soure. Cells were grown for 24 h on surose, harvested, and suspended in oleate-tergitol-ontaining medium. After further growth for the time periods indiated, ells were harvested and disrupted as desribed in the text. Enzyme ativities were measured in rude extrats. Symbols: 0, ayl-coa dehydrogenase; 0, enoyl-coa hydratase; V, 3-hydroxyayl-CoA dehydrogenase; A, 3-oxoayl- CoA thiolase; O, isoitrate lyase; V, fumarase; A, atalase. Surose density entrifugation. A 3-ml volume of the rude extrat was layered on top of a linear gradient of 30 to 60% surose (in 10 mm Triine [ph 7.4], 1 mm EDTA; volume, 30 ml). The surose gradient was entrifuged for 90 min at 20,000 rpm (48,000 x g) at 4TC in a Sorvall RC2B entrifuge equipped with a rate ontrol unit, using an SS90 vertial rotor. Frations of 1 ml were olleted from the bottom of the tube and analyzed for various enzyme ativities. The surose density in eah fration was determnined refratometrially. Enzyme assays. Ayl-CoA dehydrogenase (EC ) (11), ayl-coa oxidase (EC ) (9), etoyl-coa hydratase (EC ) (13), 3-hydroxyayl-CoA dehydrogenase (EC ) (10), 3-oxoayl-CoA thiolase (aetyl-coa ayltransferase; EC ) (23), atalase (EC ) (1), fumarase (fumarate hydratase; EC ) (3), and urate oxidase (EC ) (26) were assayed by established proedures. Determinations of isoitrate lyase (EC ) and malate synthase (EC ) were performed essentially as desribed by Dixon and Komberg (8), with slight modifiations as follows. For isoitrate lyase, the assay mixture ontained, in a total volume of 1 ml, 50 mm potassium phosphate buffer (ph 7.4), 5 mm phenylhydrazine hydrohloride, 5 mm MgCl2, 2 mm dithioerythritol, and 10 to 50,ul of enzyme solution. The reation was started by adding 6.25 mm DL-isoitrate. The inrease of absorbane at 324 nm was determined at 250C. For malate synthase the onentration of aetyl-coa was 0.1 mm and that of glyoxylate was 5 mm. All p-oxidation enzymes were assayed by using the respetive CoA esters with a hain length of 10 J. BACTERIOL. arbon atoms. Enzyme units were expressed as 1,umol of substrate used or produt formed per min. Protein ontent. Protein was assayed by the method of Bradford (5); bovine plasma gamma globulin served as a standard. RESULTS Hyphae of. rassa were grown in the presene of either 2% surose or 1 mm oleate.. rassa is able to use oleate as sole arbon and energy soure, suggesting the existane of a P-oxidation pathway in this organism. This result onfirmed the observation made by Hii and Courtright (18). In omparison to the growth of. rassa on 2% surose, the myelial yield obtained from a medium ontaining 1 mm oleate was extremely low. In this respet, the filamentous fungus. rassa behaves differently than the yeast Candida tropialis. Cultures of the latter organism grow on oleate to about the same ell mass as on gluose. To obtain higher myelial yields for ells having been exposed to oleate, we hose to grow ultures of. rassa first on surose and subsequently to shift the ells to an oleate-ontaining medium. All four enzymes of the P- oxidation sequene and isoitrate lyase, a key enzyme of the glyoxylate yle, showed a rapid inrease in ativity in response to this hange of arbon soure (Fig. 1). The time ourse of the indution for all of these enzymes was similar, reahing a maximum after about 12 h. In ontrast, atalase was hardly indued at all by the shift to oleate, and the inrease of fumarase ativity was only twofold. It is noteworthy that no ayl-coa oxidase ativity but instead an ayl-coa dehydrogenase ativity was deteted in all rude extrats (Table 1, Fig. 1). Table 1 presents the speifi ativities of various enzymes measured in extrats from ells grown on 2% surose, 40 mm aetate, or 1 mm oleate as sole arbon soures. In addition, the enzyme ativities are given in relation to the basal ativities determined in extrats from ells grown on 2% surose (indution fator). The data suggest that the fatty aid degradation pathway is under different ontrol than the glyoxylate bypass. Although isoitrate lyase was indued to the same extent by both 40 mm aetate and 1 mm oleate, the ativities of the four,b-oxidation enzymes behaved differently. The inrease in these ativities on oleate was four to five times higher as ompared to aetate. Our results related to indution are in ontrast to those reported by Hii and Courtright (18). They observed no enhanemnent by aetate of the ativities of p-oxidation enzymes; oleate yielded a signifiant indution for only one of these enzymes, 3-hydroxyayl-CoA dehydrogenase. A possible reason for these differenes might be the use of different substrates in the enzyme assays. Whereas Hii and Courtright used substrates with either 4 or 16 arbon atoms, in the present study, only substrates with a hain length of 10 arbon atoms were employed. A twofold inrease of the ativity of the mitohondrial marker enzyme fumarase was observed in the shifts to oleate and to aetate. It appears as though this enzyme exhibited a behavior very similar to that reently desribed for another mitohondrial enzyme, itrate synthase, in response to a shift to aetate (7). Intraellular loalization. To establish the intraellular loalization of the fatty aid degradation pathway in. rassa, rude extrats from ells grown on different arbon soures were subjeted to density gradient entrifugation on a surose gradient ranging from 30 to 60% surose. I- Oxidation enzymes from extrats of ells grown on either 40

3 VOL. V-OXIDATIO 161, 1985 I. CRASSA 155 TABLE 1. Effet of arbon soure in the growth medium on the ativities of various enzymes' Enzyme ativity in ells grown on: Enzyme 2% surose 40 mm aetate 1 mm oleate U/mg IFb U/mg IF U/mg IF Ayl-CoA dehydrogenase Enoyl-CoA hydratase Hydroxylayl-CoA dehydrogenase Oxoayl-CoA thiolase Isoitrate lyase Fumarase Catalase Ayl-CoA oxidase DC D D D D D a Cells were first grown for 24 h in a medium ontaining 2% surose, harvested, and grown for additional 12 h in a medium ontaining the arbon soure indiated. b IF, Indution fator. D, ot deteted. mm aetate or 1 mm oleate ompletely osedimented with the key enzymes of the glyoxylate yle, isoitrate lyase and malate synthase (Fig. 2A and B). Catalase and fumarase were learly separated and banded at densities of 1.26 and 1.19 g/m3, respetively. The separation of the glyoxylate bypass enzymes from fumarase and atalase onfirm results published by Theimer and o-workers (27, 30). Citrate synthase, a mitohondrial matrix enzyme in. rassa (7), osedimented with fumarase (data not shown). As in rude extrats, no ayl-coa oxidase ativity but rather ayl-coa dehydrogenase ativity was observed. Urate oxidase also was not deteted in frations ontaining the glyoxylate bypass enzymes (data not shown). 0. A Z B E E 7= j 0.2d-il12 1 Top 20 W1 *, ayl-ca 0.21 y Q20 na O, Lr S_O 15 2 j lo meiu an weesbeunl0hfedt eimnann Fration number FIG. 2. Distribution of various enzyme ativities in 30-to-60% surose density gradients after entrifugation of rude extrats of. rassa ells. Cells were grown for 24 h in a 2% surose-ontaining medium and were subsequently shifted to a medium ontaining either (A) 40 mm aetate or (B) 1 mm oleate-1% Tergitol for an additional 12 h. Top frations of the gradients are omitted. Symbols: 0, ayl-coa dehydrogenase; 0, enoyl-coa hydratase; V, 3- hydroxyayl-coa dehydrogenase; A, 3-oxoayl-CoA thiolase; 0, isoitrate lyase; *, malate synthase; V, fumarase; A, atalase. 1.0 In ontrast to the ativities of the other 3-oxidation enzymes, enoyl-coa hydratase ativity exhibited a dual distribution (Fig. 2A and B). In addition to the ativity peak osedimenting with the enzymes of the glyoxylate bypass, a seond peak was observed whih osedimented with the mitohondrial marker, fumarase. This result suggests the existene of at least two enoyl-coa hydratases in. rassa, one as part of the 13-oxidation system and the other loalized in mitohondria. This onlusion is further supported by the observation of relatively high speifi ativity of enoyl-coa hydratase in rude extrats from ells grown on surose (Table 1). Furthermore, when this rude extrat was entrifuged to equilibrium on a ontinuous surose gradient, enoyl-coa hydratase ativity showed the same profile as that of fumarase (Fig. 3). This result indiated that the enoyl-coa hydratase ativity is restrited to mitohondria in ells grown on 2% surose. An ayl-coa dehydrogenase EU) CD E Fration number en E0-4- Ln 0) C) FIG. 3. Distribution of enoyl-coa hydratase ativity in a 30-to- 60%o surose density gradient after entrifugation of a rude extrat of. rassa ells. Cells were grown for 24 h in a 2% surose-ontaining medium and were subsequently shifted to fresh medium ontaining the same arbon soure. Top frations from the gradient are omitted. Symbols: 0, enoyl-coa hydratase; V, fumarase; A, atalase.

4 156 KIOKA AD KUAU TABLE 2. Distribution of total enzyme ativities (rude extrats) from aetate-grown ells between soluble and partiulate frations in surose density gradients Enzyme ativitya Enzyme Mitohon- Glyoxyso- Soluble drialb malb Ayl-CoA dehydrogenase DC Enoyl-CoA hydratase Hydroxyayl-CoA D dehydrogenase 3-Oxoayl-CoA thiolase D Isoitrate lyase D Fumarase D a Enzyme ativities reovered in the different parts of the surose gradients are expressed as a perentage of the ativities (rude extrats) applied to the gradients. b Partiulate frations were designated as mitohondrial or glyoxysomal on the basis of their ontent of marker enzymes. D, ot deteted. ativity was found in the glyoxysome-ontaining frations of the surose density gradient, whereas ayl-coa oxidase ativity ould not be deteted throughout the gradient (Fig. 2). Urate oxidase was also not deteted in frations ontaining the glyoxysome-like mirobodies (data not shown). In all experiments throughout this study, various portions of all enzyme ativities measured were found to be loated in the top frations of the surose gradients after entrifugation to equilibrium. Distribution of the enzyme ativities between the partiulate and soluble frations tended to be dependent on the onditions of grinding for mehanial breakage of the ells and on the arbon soure of the growth media. In rude extrats obtained from ells grown on oleate, the soluble portions were always larger than those in extrats from aetate-grown ells. Distribution of enzyme ativities from aetate-grown ells (rude extrats) between soluble and partiulate frations of surose gradients are given in Table 2. Major portions of all individual p-oxidation ativities and marker enzymes were reovered in a single partiulate peak (glyoxysomal or mitohondrial), and these portions were always larger than the respetive ativities in the soluble frations. Considering the mehanial and osmoti fragility of mirobodies, this result was taken as an indiation that fatty aid degradation is truly sequestered within glyoxysome-like mirobodies and that the soluble portions of the various enzyme ativities reflet rupture of the respetive ell organelles. This assumption is supported by the observation that prolonged grinding as well as the appliation of stronger mehanial fore for ell breakage onsiderably inreased the soluble portion of the different ativities. DISCUSSIO The data presented here show the existene of an induible non-mitohondrial p-oxidation system in. rassa. This onlusion is based on the detetion of all individual enzymes of the p-oxidation sequene in ell-free extrats (Fig. 1), on the different amounts of these ativities in rude extrats from ells grown on different arbon soures (Table 1), and on the lear separation of these ativities from mitohondrial marker enzymes on surose density gradients (Fig. 2). The organelles housing the p-oxidation system annot onlusively be identified as glyoxysomes solely on the basis of their ontent of glyoxylate bypass enzymes (Fig. 2). Results from Theimer and o-workers (27, 30), however, who determined enzyme ontent as well as morphologial J. BACTERIOL. properties, strongly suggest that the organelles from. rassa that band at a density of 1.21 g/m3 are glyoxysomelike mirobodies. This glyoxysome-like mirobody population of. rassa has two remarkable properties. First, it does not ontain atalase (Fig. 2), although in general this enzyme is ompartmentalized in mirobodies. For. rassa, Theimer and o-workers (27, 30) provided evidene of a seond mirobody population (1.26 g/m3), whih by both mirosopi and biohemial riteria appeared to be peroxisomes. Seond, the glyoxysome-like mirobodies of. rassa lak ayl-coa oxidase ativity and instead ontain ayl-coa dehydrogenase ativity. This is a surprising result beause ayl-coa oxidase is assumed to be a general enzyme of p-oxidation in mirobodies, whereas ayl-coa dehydrogenase is regarded as its ounterpart in mitohondrial P- oxidation (17). There is only one other report (15) thatmentions the presene of ayl-coa dehydrogenase ativity in extrats of Euglena grailis and haraterizes its P- oxidation system as being loated in glyoxysomes. In that ase, however, two atalase loations were deteted, namely, in glyoxysomes and in partiles banding in a surose gradient at a density of 1.26 g/m3. The loalization of the p-oxidation pathway in glyoxysomes without peroxisomal funtion is different from all results so far reported for non-mitohondrial fatty aid degradation in higher animals (14, 20, 21), higher plants (2, 6, 14, 28), and yeasts (25). On the other hand, information about p-oxidation in euaryoti miroorganisms, espeially fungi (22), is so sare that onsiderable work is needed to deide whether the separation of p-oxidation and H202 metabolism in different mirobody populations is restrited to ertain fungi or even only to. rassa. Enoyl-CoA hydratase is the only p-oxidation enzyme whih was not exlusively loated in the glyoxysome-like mirobodies but was also deteted in mitohondria (Fig. 2 and 3). The same dual distribution of this enzyme ativity has been reported reently for another filamentous fungus, Aspergillus tamarii (22). The absene of the other P- oxidation enzymes from mitohondria in. rassa suggests that the mitohondrial enoyl-coa hydratase may be involved in a different metaboli pathway. Therefore, the demonstration of enoyl-coa hydratase ativity alone is not a reliable indiation of the presene of fatty aid degradation. The results of these studies leave a number of questions open. Among those are the nature of the hydrogen aeptor of the apparently glyoxysomal ayl-coa dehydrogenase and the relationship of this enzyme to the well-haraterized mitohondrial ayl-coa dehydrogenases (12). Sine the ayl- CoA dehydrogenase from. rassa is urrently being purified in our laboratory, larifiation of the questions should soon be possible. Sine mitohondrial and peroxisomal p-oxidation enzymes differ distintively (17), strutural haraterization of all p-oxidation enzymes from. rassa should be espeially interesting with regard to their resemblanes to the two systems. ACKOWLEDGMETS This work was supported by the Fonds der Chemishen Industrie. LITERATURE CITED 1. Aebi, H Katalase, p In H. U. Bergmeyer (ed.), Methoden der Enzymatishen Analyse, 3rd ed. Verlag Chemie, Weinheim, Federal Republi of Germany. 2. Beevers, H Mirobodies in higher plants. Annu. Rev.

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