Balinska t't af.: Folate metaholism In Aspergillus

Pteridines Vol. 4, 1993. pp. 56-59

Regulation of Folate Metabolizing Enzymes In Fungus Aspergillus nidulans*

Malgorzata Balinska # §. Renata Natorfft and Andrzej Paszewski t

i' M . Nencki Institute of Experimental Biology, Polish Acallemy of Sciences. Warsaw. Poland t Institute of Biochemistry and Biophysics. Polish Academy of Sciences. Warsaw. Poland.

(Rcl:civcd January 30. 1993)

Summary

The regulatio n o f nwthionine sy nthase. serine hydroxymethyltransferase. methylenetetrahydrofolate ()xido­ relluctase anll dihydrofolate reductase in A.\j1crgil/lls lIi(/lIlollS have heen found to he controlled hy endoge­ nous pools methionine and homocysteine. Mutants impaired in sulfur metaholite repression. which have highly elevated pools of these amino acids. also show elevated levels of rolate metaholi zi ng enzymes mentio­ ned ahove. This eftect was tound to he the result of the accumulation of endogenous homocysteine. which induces fiJlate enzymes. High concentration (up to 5 mM) or methionine in growth medium leads to repression of these enzymes. It appears. therefore. that the levels of folate metaholizing enzymes arc determi­ ned hy the ratio of cellular levels or methionine a nd homocysteine. Key words: Methionine synthase. Serine hydroxymethyltransferase. Dihydroiolate reductase. Methylenetetrah\drofo late oxidoreductase. A . ~pergil/us

Introduction {Ins. These genes control sulfur metabolite repres­ In many organisms (inculuding fungi, higher sion - a system which shuts off metabolic pathways plants, mammals) homocysteine serves as a precursor needed ror utilization of poor sulfur sources like of methionine (I). Since fol ate metabolism provides sulfate when a prererahle source such as methionine a methyl group for methylation of homcysteine hy is avaiable. In the mutant strains an increased pool methio nine synthase. a proportion of intracellular of homocysteine leads to elevation of the level of level of both amino acids can regulate folate meta­ folate metabolizing enzymes. while the additon o f holism as well as sulfur amino acid synthesis (2). methionine causes repression of these enzymes. It has been known that high endogenous concentra­ tion of methionine is effective in reducing the level of folate enzymes. intracellular pools of folate and Materials and Methods anti folate as well as levels of some coordinately re­ gulated enzymes involved in amino acid metabolism MutagenesL\' and selection of reKU/(/fory mU/ullls (3.4). In this paper we present data on the regulation Mutations in presumptive sulfur regulatory genes of folate enzymes by mutations in Aspergillus lIidul- were isolated a~ suppressors of the metA / 7 mutation affecting cYSlaliolline y-synthase (5) following ultra­ § Author to \vholll corn:sronckncl' .should be addrcssed. *This anick was prl'sentell at I ill' Si\lh Inil'mal io nal Cllnfcr­ violet irradiat io n or conidia or II/('[A 17 strain at ellcc Oil I'teridin," alld RcklL'd Bi ogl'llic .'\mille" dlltl 1·" lall·s. 1-5 percent suryi\"a l leve l. For the idcnlitiL'atioll o r h,'leI JUlll' I,)'):' ill Sl'lHiI "-"rca. thl'-;e mutants the standard proL'l'durL' using propa r-

Pl criel illl'S ! Vo l. -I ; No. I Balinska et al.: Folate metabolism in Aspergillus 57

gylglycine (6) was employed to distinguish them ted. Strains carrying these mutations show derepres­ from other types of suppresors. Details concerning sion of several enzymes involved in synthesis of sul­ mutant isolation will be described elsewhere (71. fur amino acids (Notorff R, Balinska M. & Paszew­ ski A.: upublished results). Wedia. culture conditions and enzyme assays For further characterization of scon mutants the pools of methionine and homocysteine in mycelia Liquid minimal medium described previously grown in the presence of 35S-sulfate were determin­ with appropriate supplements was used (8). Mycelia ed (Fig. I). It is evident that all scon mutants show were harvested, collected and cell-free extracts pre­ higher pools of these amino acids as compared with pared using standard procedure (3). the wild type strain. What is more striking, the addi­ Methionine synthase was assayed as described pre­ tion of methionine to the growth medium has no \iously (4), serine hydroxymethyltransferase and me­ or very little effect on sulfate assimilation in the thylene oxidoreductase according to Scrimegour and mutants in contrast to the wild-type strain in which H uennekens (8) and dihydrofolate reductase as des­ the sulfate assimilation is almost eliminated. The .:ribed by Hanggi and Littlefield (9). levels of methionine and homocysteine pools in the Protein was estimated by method of Bradford (10) mutants are also less affected by exogenous methio­ with bovine serum albumin (fraction V) as a stan­ nine than in the wild-type strain. The double mutant dard. sconB9sconC3 shows highest pools with predomin­ ance of methionine. Dl:'tl:'rmination of pools of 35S-labelled compoundl' The results presented on Figs. 2 and 3 indicate that the levels of folate metabolizing enzymes are Cultures were grown in mimi mal medium supple­ elevated in scon mutants as compared to the wild mented with 2 mM"S-sulfate under standard con­ type strain. The highest level of the folate enzymes dition for 18 h. Mycelia were harvested and 3'S-label­ was observed in the double mutant sconB9sconC3 led compounds isolated as described previously (Fig. 3) and in the \\ild type strain grown on homo­ (7. II). .:ystei ne (Fig. ~). Exogenous methionine represses synthesis of these enzyme" both in the mutants and in wild type strain Results and Discussion (Figs. ~ and 3). It appears therefore that synthesis of folate enzymes is induced by homqcysteine and Mutations in t!lree regulator: genes sconB. sconC repressed by methionine so their levels are determin­ and sconD suppressing IIll:'L417 mutations were isola- ed by relative pools of these amino acids. Evidently

50

40

~ >- U 30 '".. 0 20 E ::t

10 m

_.m +m +m 0 l WT scon 62 Lscon C3

Figure I. Accumulation of "S-Iabelled methionine and homocysteine in mycelia of the 'COil and "ild type (WT) strains in the presence o[ " SO. -, and absence or presence (t- m) of 5 mM methionine. ~ total accumulation (" S retained on Dowex 50 (H t )). [ 'j homocysteine, • methionine. Mycelia were grown in the presence of methionine tor 6 h. then Na2"S04 (4X 10' cpm/ mmol) was added for next 12 h. Detennination of methionine and homocysteine in mycelial extracts is described in Materials and Methods.

Pteridines / Vol. 4 / No. I 58 Balinska ef of.: Folate metabolism in Aspergillus

100 in the scan mutants the inductive effect of homocys­ teine overcomes repression exerted by methionine. 80 A similar mode of regulation has been described

+h for BITindependent methionine synthase in E. coli (13.14). On the other hand exogenous methionine 60 caused a decrease of 5-methyltetrahydrofolate (subst­ ,.. .:; rate of methionine synthase) and shift of folate pool +h ~ « 40 from 5-methyltetrahydrofolate to tetrahydrofolate. It was also observed that S-adenosylmethionine repres­ sed methylenetetrahydrofolate oxidoreductase. Ho­ 20 mocysteine diminish these effects (3.15). These data suggest that observed effect may be attlibuted not only to methionine-mediated repression of folate en­ o zymes. but also to changing of intracellular pools Figure 2. Activity of folate metabolizing enzymes in wild of folate coenzymes. strdin cultured in absence and presence of 5 mM methionine ( + m) or 3 mM homex:ysteine ( t h). ~ dihydrofolate reductase. :··i serine hydroxymethyhra nsferasc. • methykncterahydrofo late oxidoreductase. ill methionine synthase. Gro\\lh conditions. Acknowledgements mycelial extracts preparation ami enzyme assays were as desc­ rihed in Materials and methods. Methionine or homocysteine This work was partially funded by State Commit­ \\'as added at the sta n o f culture. tee of Scientific grant number 2231 /4/91.

100

80

60 >- ? u « 40

20

0 scan 82 s(;an 89 scan C3 scan 89 scan C3

Figure 3. Activity of folate metabolizing enzymes in scon strains cultured in absence and presence of 5 mM methionine ( + m). Ii!! dihydrofolate reductase. H serine hydroxymethyltransferase. • methyleneterahydrofolate oxidoreductase. a methionine synthase. Growth conditions. mycelial extract preparation and enzyme assays as described in Materials and methods. Methionine was added at the start of culture.

References 4. Paszewski A. Prazmo W. Landman-Balinska M. Regula­ tion of homocystei ne metabolizing enzymes in A.lpl'rgillus I. Flavin M. In: Metabolism of sulfur compounds. vol VII nidlilans. Mol Gen Genetics 1977: 155: 109-112. New York: Academic Press. 1975: 457-503. 5. Cybis J. NatorfT R. Lewandowska I. Prazmo W. Pas7ewski 2. Nadolska-Lutyk J. Balinska M. Paszewski A. Interrelated A. Mutations alkcting cysteine synthesis in A,lpergilllls ni­ regulation of sulphur containing amino acid biosynthetic dlilans: characwrization and chromosome mapping. Gell enzymes and folate-metabolizing enzymes in Alpelxillus lIi­ Res 19::;::;: "1' X5 -XX . dlilans. Eur. J. Biochem. 19X9: IX!: 231-2.l". 6. PasLc\\,ki A Grabski J. Enzymatic lessions in methionine 3. Rhee MS. 10hnson TR. Priest LJG . Galivan J. The cOcct mutant' of .-tIP£'lXillIIS nidlilans: role and regulation of a lt er­ of methionine on methotrexate metabolism in rat hepato­ nati\c path\\'ay lor cysteine and methionine sy nthesis. J cytes in monolaye r culnlre. Biochim Biophys Acta 19X9: B'lc'tcriol 1975: 124: l\9~-904 . I 0 I I: 122-1 2R - "," 'l to rl1' R. Balil1ska M. P'lqc",ki \ . '\1 least four regula-

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tory genes control sulphate metabolism repression in Asper­ 12. Paszewski A Praimo W. Nadolska J. Regulski M. Muta­ gillus nidulans. Mol Gen Genetics (in press). 1993. tions affecting the sulfur assimilation pathway in Aspergil­ 8. Paszewski A Grabski J. Regfulation of S-amino acids bio­ lus nidulans: their effect on sulfur amino acids metaholism. synthesis in A5pergillus nidulans. Mol Gen Genetics 1974: J Gen Microbiol 1984: 130: 1113-1121. 135: 307-320. 13. Cai X-Yo Redfield B. Maxon M. Weissbach H. Brot N. 9. Scrimegour KG, Huennekens FM. In: Bums FM. eds. Hand­ The effect of homocysteine on melR regulation of melR buch der Physiologisch und Pathologisch Chemischen melE and metH expression in vitro. Biochem Biophys Res Analytik. Springer Verlag. Berlin. 1966: 181-208. Commun 1989: 163: 79-83. 10. Hanggi VJ. Littlefield JW. Isolation and characterization 14. Urbanowski ML. Stauffer GV. Role of homocysteine in on the multiple forms of the dihydrofolate reductase from metR-mediated activation of metE and merH genes in Sal­ methotrexate resistant hamster cells. J Bioi Chern 1974; monel/a typhimurium a nd Escherichia coli. J Bacteriol 1989: 249: 1390-1397. 17 1: 3277-3281. II. Bradford MM. A rapid and sensitive method for the quan­ 15. Kutzhach CA Stokstad ELR. Feedback inhibition of me­ titation of microgram quantities of protein utlizing the thylenetetrahydrofolate reductase in rat liver by S-adenosyl­ principle of protein-dye binding. Anal Biochem 1976; 72: methionine. Biochim Biophys Acta 1967; 139: 217-220. 248-254

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