Significance of the Non-Oxidative Pentose Phosphate Pathway In
Total Page：16
File Type：pdf, Size：1020Kb
Significance of the Non-Oxidative Pentose Phosphate Pathway in Aspergillus oryzae Grown on Different Carbon Sources Maria Luisa Peleatoa, Teresa Muiño-Blancob, José Alvaro Cebrian Pérezb, and Manuel José López-Pérezb Departamento de Bioquimica y Biologia Molecular y Celular. a Facultad de Ciencias, b Facultad de Veterinaria, 50009 Zaragoza, Spain Z. Naturforsch. 46c, 223-227 (1991); received September 27, 1990/January 25, 1991 Aspergillus, Pentose Phosphate Pathway, Fungal Development Specific enzyme activities of the non-oxidative pentose phosphate pathway in Aspergillus oryzae mycelia grown on different carbon sources were determined. Mycelia grown on glu cose, mannitol and ribose show the highest specific activities, ribose 5-phosphate isomerase being specially very enhanced. Moreover, transketolase, transaldolase, ribose 5-phosphate iso merase and ribulose 5-phosphate 3-epimerase were determined in different developmental stages of mycelia grown on glucose, mannitol and ribose. The non-oxidative pentose phos phate pathway is more active during conidiogenesis, except for ribulose 5-phosphate 3-epimerase, suggesting a fundamental role of this pathway during that stage to supply pen toses for nucleic acids biosynthesis. A general decrease of the enzyme activities was found in sporulated mycelia. Arabinose 5-phosphate was tested as metabolite of the pentose pathway. This pentose phosphate was not converted into hexose phosphates or triose phosphates and inhibits significantly the ribose 5-phosphate utilization, being therefore unappropriate to sup port the Aspergillus oryzae growth. Introduction of the pathway, with the exception of epimerase, The Aspergilli use a wide range of organic com have been reported in Candida utilis [8] and in pounds as source of carbon and energy, and the A. nidulans [9] in which the specific activities and growth yield is quite different for hexoses, pen content of transaldolase isoenzymes of various toses and pentitols [1], Many of them support strains were also reported [10]. growth, after an obvious time lag, revealing an The pentose phosphate pathway plays different adaptive metabolism to particular environmental roles during Aspergillus differentiation, and one of conditions. The differences in growth on different the main changes in enzyme profiles, associated carbohydrates could be due to regulation of key with conidiogenesis in filamentous fungi, is the catabolic enzymes or to specific uptake systems stimulation of this pathway. The increased capaci [2-4]. ty of the pathway is consistent with an increased Fungi generally utilize aldo-pentoses by reduc demand of NADPH for biosynthesis. tion to pentitols, which are interconverted through In fungi, most of the studies on the interference keto-pentoses and other pentitols to D-xylulose, of different nutritional conditions with the pentose the only phosphorylated pentose to enter the pen phosphate pathway basically concern the oxida tose phosphate pathway, as was proposed by tive phase, and especially glucose 6-phosphate de Lewis and Smith [5] and confirmed by Hankinson hydrogenase [11, 12], This step, together with the transketolase activity are the two principal en [6] in Aspergillus nidulans. The occurrence of the pentose phosphate path zymes controlling the two branches of the path way in filamentous fungi, including species of way. The control of the non-oxidative branch at the transketolase step has been less studied and, to Aspergillus, was documented by Blumenthal in date, there have been no reports of allosteric regu 1965 [7]. The specific activities of all the enzymes lation of transketolase or transaldolase. This paper is intended to provide information on the four enzymes of the non-oxidative branch Reprint requests to M. J. Lopez-Perez. of the pentose phosphate pathway during the Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen growth of Aspergillus oryzae on different carbon 0939-5075/91/0300-0223 $01.30/0 sources. 224 M. L. Peleato et al. ■ Non-Oxidative Pentose Phosphate Pathway Materials and Methods a-glycerol phosphate dehydrogenase and 0.1 m M Enzymes and chemicals NADH. A suitable dilution of the enzyme prepa ration was added. Distilled water was added to a Enzymes, sugar phosphates and coenzymes final volume of 1 ml after the addition of the were obtained from Sigma Chemical Co. (St. sample. The oxidation of NADH was followed at Louis, Mo., U.S.A.). All other chemicals and 340 nm. solvents were of analytical reagent grade. Aldolase (EC 4.1.2.13) was assayed by the method described by Hankinson and Cove [9] for Growth of mycelium aldolase in Aspergillus nidulans. The Aspergilli were grown with different sub Triose and hexose phosphate formation from strates as sole carbon source, as previously de pentose phosphate was measured with the method scribed [12]. Conidial suspensions were prepared described by Williams et al. [16] based on Horek- in sterile distilled water. A small volume of the ker et al. [17]. Pentose phosphates were incubated concentrated conidial suspension was added to with dialyzed preparations of Aspergillus oryzae 100 ml of Mulder’s medium in a Roux flask. The and, at different incubation times, aliquots were flasks were maintained without rotation and the withdrawn, and triose phosphate and hexose phos temperature was controlled at 30 °C. The mycelia phate formation were measured. were harvested, washed and stored at -20 °C for 1-2 weeks. Crude extracts were prepared using a Protein determination glass homogenizer. The protein measurement was performed using the method described by Lowry et al. [18]. Analytical procedure Enzyme assays. All enzyme assays were per formed at 25 °C. The assay for transketolase was Results performed as described by Simcox et al. [13]. Table I shows the enzyme activities of the non- Transaldolase was assayed as described by Novello oxidative pentose phosphate pathway determined and McLean [14], For ribose 5-phosphate iso- in crude extracts prepared from mycelia of Asper merase and ribulose 5-phosphate 3-epimerase, the gillus oryzae grown on different carbon sources assays proposed by Kiely et al. [15] were used. and collected at the vegetative growth phase. A possible arabinose 5-phosphate 2-epimerase Ribose 5-phosphate isomerase and ribulose was tested using the following incubation mixture: 5-phosphate 3-epimerase, when detected, showed 40 m M glycylglycine/KOH, pH 7.4; 0.6 m M arabi higher specific activities than transketolase and nose 5-phosphate; 0.13 m M xylulose 5-phosphate; transaldolase. Glucose and mannitol-grown myce 20 |aM TPP; 5 m M MgCl2; 0.3 units transketolase; lia presented very similar specific activities for all 4 units triose phosphate isomerase; 1 unit enzymes assayed. Table I. Specific enzyme activities of the non-oxidative pentose phosphate pathway in mycelia grown in different carbon sources. Aspergillus oryzae was grown in 6% (w/v) carbon source, and enzyme activities were determined in the crude extracts. The results are means of three determinations and the standard deviations were less than 10%. Transketolase Transaldolase R5P isomerase Ru5P 3-epimerase [mU/mg] [mU/mg] [mU/mg] [mU/mg] Glucose 111 200 1.105 369 Mannitol 98 227 1205 365 Ribose 227 312 788 367 Arabinose 104 95 250 122 Fructose 52 272 n.d. n.d. Sorbitol 164 120 n.d. n.d. n.d., not detected. M. L. Peleato et al. ■ Non-Oxidative Pentose Phosphate Pathway 225 However, Aspergillus oryzae grown on ribose 0,12 shows a decrease of the ribose 5-phosphate iso- o E 0,10 merase activity whereas the transketolase and E transaldolase increased their activity in compari a> 0,08 N to son with those of the glucose or mannitol-grown .c mycelia. Growth on arabinose causes a relative a. 0,06 oV) decrease of almost all enzyme activities. When 0,04 growth was carried out in the presence of either / * N fructose or sorbitol, neither ribose 5-phosphate 0,02 isomerase nor ribulose 5-phosphate 3-epimerase 0,00 n ti^ A-4 |--------. - * ,----------------+ activities were detected. 200 400 600 17h In order to obtain further information on the Time (min) activities found when the growth occurred in ara- binose-containing media, a series of experiments Fig. 2. Triose phosphate formation by extracts of myce lium grown on ribose-containing media as carbon source; was carried out in which pentose-dependent the extracts were incubated in the presence of 2 (imol of hexose and triose phosphates formation by ribose- ribose 5-phosphate (•), ribose 5-phosphate and arabi grown mycelium extracts were studied. The results nose 5-phosphate (■), arabinose 5-phosphate (A). obtained are shown in Fig. 1 and 2. Ribose 5-phosphate was converted into hexose and triose phosphates but arabinose 5-phosphate was not. phate formation from ribose 5-phosphate is 2 ^im When both pentose phosphates were present in the and 64 |im respectively. Neither hexose phosphates incubation mixture the ribose-dependent hexose nor triose phosphates formation were detected phosphates formation was also significantly inhib when only arabinose 5-phosphate was present in ited. However, arabinose only slightly inhibited the incubation mixture. When mycelia grown on formation of triose phosphates from ribose, and in arabinose were used, very small amounts of hexose fact a tendency to accumulation was observed at phosphates and triose phosphates were detected as longer incubation periods (Fig. 2). The triose and formed from ribose phosphate. When arabinose hexose formation from the mentioned pentose 5-phosphate was present, neither triose phos phosphates were also measured in crude extracts phates nor hexose phosphates were found at all. from mycelia grown on arabinose as sole carbon Finally, the specific activities of the non-oxida- source. The maximum hexose and triose phos- tive pentose phosphate pathway enzymes were studied during the fungal development in the pres ence of the best three carbon sources which induce the non-oxidative pentose phosphate pathway o E activities (glucose, mannitol and ribose). Three E, stages were considered: vegetative growth, coni- a> dial differentiation (conidiogenesis) and fungal szCB spore formation.