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Aldosugar dehydrogenases from Neurospora crassa Partial purification and characterization of D-arabinose: NAD dehydrogenase
Volum~ 30, number. I
FEBS LETCI'ERS
ALDOSUGAR DEHYDROGENASES Partia| purification :rod characterization
February 1973
FROM NEUROSPORA CRASSA of D-arabinose: NAD dehydrogenase
G. P I N C H E I R A , G. LEON and T. U R E T A * LJel~rramento de Bioqu(mica. Factdtad d,-" MIedic'zrta and Oetrartamento de Biolog~a, Faculrad dc Ciencias. Utffverzidad de Chile, Casilla 6671, Santiago-4, 6?tile
Received 4 December 1972
1. Introduction The py~,dine nucleotide-linked o x i d a t i o n o f free sugars t o th~tr c o r r e s p o n d i n g lactones has been described in several organisms [ 1 - 1 4 ] . T h e e n z y m e s acting o n free a l d o p e n t o s e s seem to catalyze the first reaction o f a p a t h w a y leading to intermediates o f the citrtc acid cycle [31 b u t the possibility o f their involvement in b i o s y n t h e t i c p a t h w a y s should n o t be lightly dismissed. Despite their general o c c u r r e n c e [t5] only the arabinose d e h y d r o g e n a s e s from P s e u d o monas [3, 5--7] and a related e n z y m e f r o m mammalian liver [! i - - 1 3 ] have been studied with some detail. We wish now t o r e p o r t the presence in N e u r o s p o r a crassa of several sugar dehyd~togenases, anti the partial purification and p r o p e r t i e s o f a D-arabin¢,se:NAD d e h y d r o ~nase.
2. Methods Wild t y p e strain 7 4 A o f N e ~ r o s p o r a crassa was grown in V o g d ' s m e d i u m [ ! 6 1 containing 2% su,~rose as carbon source. T h e flasks were shaken o n an Eberbach r o t a t o r y shaker at a b o u t 140 rpm at 2 i~°. Approx. 8 g ( w e t weight) o f myeelia were suspetFted in ice-chilled O 05 M Tris-HC! p H 7.2 b u f f e r and grinded in an ice chilled mortar. T h e suspension ~ a s centrifuged at 4 ° at 4 , 0 0 0 r p m for 2 0 rain in a Sorvall RC 2 centrifuge. T h e s u p e m a t a n t liquid was f u r t h e r correspbndenee to: Dr. Tito Ureta, Casilla 6671, Santiago.4, Chile.
*Addte~ al|
North-llollar~d P u b l i s h i ~ Company -- Amsterdam
centrifuged at 4 0 , 0 0 0 rpm during 60 rain in a Hitachi ultracentrifuge. T h e sediment was discarded. Dehydrogenase activity was assayed through the reduction o f NAD or NADP at 340 n m in the presence o f sugar. T h e reaction mixtures contained 10 mM KCI, ! 0 0 mM Tris HCI b u f i ~ r ( p H 7.5), 1.6 mM EDTA, O.5 mM NAD + or NADP ~, 100 mM ~ugar and e n z y m e in a final volume o f 0.5 rid. A similar system w i t h o u t sugar was used as a blank. A Gilfotd s p e c t r o p h o t o m e t e r t h e r m o s t a t t e d at 30 ° orovided with a recording a t t a c h m e n t ~,Jas used. One -auit o f activity is the a m o u n t o f e n z y m e ca*.a~yzing the r e d u c t i o n o f i /amole ot-NAD(P) + p~r rain ~t 30 °. Polyacrylamide gel electropht~resis was p e r f o r m e d at a b o u t 10 ° exactly as described b y Davis [17] at 4 mA per column. Histochemical localization o f e n z y m e activities were done b y stair:lug the gels per I0 mitt in the m e d i u m described above plus 0.04- ntg,/ml phenazhte methosulfate and 0.4 mg/ml nitroblue tetrazolium. Partial purification o f D-arabinose d e h y d r o g e n a s e was accomplished b y DEAE-cetlulose ( W h a t m a n DE-52) c h r o m a t o g r a p h y in a I X 16 cut gtass columiI. A linear gradient ( 2 4 0 ml) from 0 to 0.5 M KCi was applied to the c o l u m n and 2-ml fractions were collected in a Gilson microiYactionator. Protein was estimate d by measurit~g the absorbance o f the effluent at 280 rim.
3. Results Direct measurement o f dehydrogenase activity in high-speed supernatant liquids revealed ~outinely very 1! I
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drying ~esulted in the almost c o m p l e t e i r e s . o f the activity. Thu.s, w i t h m t t further pudfi.~, tton the fractiom were U ~ d for e n z y m e characterization., .. Substrate spec~u:ity'studies are d~own m table ! using N A D ÷ as hydro~gen accept o r i n a s m u c ~ as NADI" was c o m p ~ t e l y i,aactive. D-Arablnose is b y far the best txtbst~te, a l t h o u g h L ~ f u c o ~ and D-n'bose w e r e also a t ~ c k e d b u t to a much. lesser>ex, teni.~ The.effect o f D-.arab inose c o n c e n t r a t i o n on the velocity o f NAD.+ reduction and viceversa i s shown in fig. 3. Classical M i c h a e l i s - M e n t e n kinetics were obtained. T h e calculated K m values were 6.7 X 10 -4 M for D-azabinose and 2.96 X 10 - 4 M for N A D +. We Imve n o t p e r f o r m e d detailed analysis o f the reaction products but since t h e a d d i t i o n ofhydroxYlamine to lfle reap tion. mixture at pH 6.5 resulted m hydroxamate p r o d u c t i o n w e . p r e ~ t m e that an arabonolactone h the immediate p r o d u c t o f the o x i d a t i o n reaction. % . .
•
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Thts report adds several e n z y m e s to the grow~g, list o f dehydxogehases that c a t a l y z e oxidation-reduction reactions o.f f r e e . s u g a r . T h e e n z y m e s fr.qm N ~ are present in very small a m o u n t s . F o r instance, hex0.
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Volume 30. number 1
kinase and glucose 6-phosphate dehydrogenases are at least one thousand times more active than D-arabinose dehydrogenase. Thus, the pathways in which these delq, drogenases are involved must..bo,..of.nlinor~ ~'~an7...... :tit ative~poi~t~fi~ei~iio::iile Celli.ii!a:t::ieast£ / o m i ! ~ ::p6h~t~ o f:view 0~: energy pmdu¢tiQniHowe~t.:the:p~sSibiiity: of:their being:!inVolvedin bios~/nthetiepathwiiys llas :" n0tbeen':ruled :o~it:(see:fo~ iristanc~e::i]8]:): ( :.II: : our stt:dieSiwith D.arabinosedehydrogenasc from N. crassa reVeaL striking d i f f e r e n c e s w i t h o t h e r . D.arabinoseldehydr0genases sO far:: reported: For instance:, the enzyme:fromrs~aomo~ [6] .is able to utilize both NAD*.-and NADP+.; Ais0, the JVeurospora dehydrogenase .is different from the.pig enzyme whose preferred substrata is L-fucose [ 12]. Finally, it is also different from the D-arabinose dehydrogenase from rat liver which is active only with NADP + as hydrogen acceptor [ 1 I, 15]. Preliminary experiments on colonial mutants of 2"Vettrospora ha:,e shown interesting quantitativo differences in the amount of arabinose d e h y d r o g e n a ~ and will be soon reported.
Acknowledgements This work was supported by grants from the Comisi6n de Energi'a Nuclear, Comisi6n Investigaci6n Cientffica, Facultad de Medicina, y Comisi6n Central de lnvestigaci6n Cientt~ca, Universidad de Chile. We thank Miss Jasna Radojkovi~ and M,ss Maria Riqttelme I'or their help in the early phases of this study.
114
'
FEBS LETTERS
February 1973
References i
[1] D.C. Harrison, Biochem. J. 25 (1931) t0t6.
4493;
-
lTl A~L~CIine andA.S.L. Hu, J. BioL Chem. 240 (i965) 4498.- :
181 P. Cuatrecasas and $. Segal° J. BioI. Chem. 241 (1966) 5904. [91 9. Cuatrecasa,~ and S. Segal, J. Biol. Chem. 241 (1966) 59~o. [1ot E. Bcutlet and M. Morfison, J. Biol. Chem. 242 (1967)
5239, l t t l R,P :~detzgerand A.N. Wick, B|ochem. B|ophys, Res. Conlmun. 26 (1967"~ 742. lt2l l-I. Seha¢lttez, J, Sarney, E.J, MeGuirc and S, Roseman, J. ~'iol, Chem. 244 (1969) 478~.
l|31 F .W. Mobley, R.P. Metzger and A.N. Wick, Arch. lllo. v l ~ n l . Biophys. 139:(1970) 83. [141 "l. Urota and $. RadoJkovi~, FEllS Lettots 9 (1970~ 57, ltSl T. Ur©ta, J. Radojkcvid and M. Rlquclme. in preparation, 1.16l ll.J Vogel, lL~ierobial Genetics Bull. l 3 (1956) 42. [17 J B.]. Davis, Ann. N.Y. Acad. Sol. 121 (1964) 4 0 4 . l~81 R.I. Katzman, E, Lisowska and R.W. Jeanloz. Bioch,,m. J. 119 (1970) 17.
FEBS LETCI'ERS
ALDOSUGAR DEHYDROGENASES Partia| purification :rod characterization
February 1973
FROM NEUROSPORA CRASSA of D-arabinose: NAD dehydrogenase
G. P I N C H E I R A , G. LEON and T. U R E T A * LJel~rramento de Bioqu(mica. Factdtad d,-" MIedic'zrta and Oetrartamento de Biolog~a, Faculrad dc Ciencias. Utffverzidad de Chile, Casilla 6671, Santiago-4, 6?tile
Received 4 December 1972
1. Introduction The py~,dine nucleotide-linked o x i d a t i o n o f free sugars t o th~tr c o r r e s p o n d i n g lactones has been described in several organisms [ 1 - 1 4 ] . T h e e n z y m e s acting o n free a l d o p e n t o s e s seem to catalyze the first reaction o f a p a t h w a y leading to intermediates o f the citrtc acid cycle [31 b u t the possibility o f their involvement in b i o s y n t h e t i c p a t h w a y s should n o t be lightly dismissed. Despite their general o c c u r r e n c e [t5] only the arabinose d e h y d r o g e n a s e s from P s e u d o monas [3, 5--7] and a related e n z y m e f r o m mammalian liver [! i - - 1 3 ] have been studied with some detail. We wish now t o r e p o r t the presence in N e u r o s p o r a crassa of several sugar dehyd~togenases, anti the partial purification and p r o p e r t i e s o f a D-arabin¢,se:NAD d e h y d r o ~nase.
2. Methods Wild t y p e strain 7 4 A o f N e ~ r o s p o r a crassa was grown in V o g d ' s m e d i u m [ ! 6 1 containing 2% su,~rose as carbon source. T h e flasks were shaken o n an Eberbach r o t a t o r y shaker at a b o u t 140 rpm at 2 i~°. Approx. 8 g ( w e t weight) o f myeelia were suspetFted in ice-chilled O 05 M Tris-HC! p H 7.2 b u f f e r and grinded in an ice chilled mortar. T h e suspension ~ a s centrifuged at 4 ° at 4 , 0 0 0 r p m for 2 0 rain in a Sorvall RC 2 centrifuge. T h e s u p e m a t a n t liquid was f u r t h e r correspbndenee to: Dr. Tito Ureta, Casilla 6671, Santiago.4, Chile.
*Addte~ al|
North-llollar~d P u b l i s h i ~ Company -- Amsterdam
centrifuged at 4 0 , 0 0 0 rpm during 60 rain in a Hitachi ultracentrifuge. T h e sediment was discarded. Dehydrogenase activity was assayed through the reduction o f NAD or NADP at 340 n m in the presence o f sugar. T h e reaction mixtures contained 10 mM KCI, ! 0 0 mM Tris HCI b u f i ~ r ( p H 7.5), 1.6 mM EDTA, O.5 mM NAD + or NADP ~, 100 mM ~ugar and e n z y m e in a final volume o f 0.5 rid. A similar system w i t h o u t sugar was used as a blank. A Gilfotd s p e c t r o p h o t o m e t e r t h e r m o s t a t t e d at 30 ° orovided with a recording a t t a c h m e n t ~,Jas used. One -auit o f activity is the a m o u n t o f e n z y m e ca*.a~yzing the r e d u c t i o n o f i /amole ot-NAD(P) + p~r rain ~t 30 °. Polyacrylamide gel electropht~resis was p e r f o r m e d at a b o u t 10 ° exactly as described b y Davis [17] at 4 mA per column. Histochemical localization o f e n z y m e activities were done b y stair:lug the gels per I0 mitt in the m e d i u m described above plus 0.04- ntg,/ml phenazhte methosulfate and 0.4 mg/ml nitroblue tetrazolium. Partial purification o f D-arabinose d e h y d r o g e n a s e was accomplished b y DEAE-cetlulose ( W h a t m a n DE-52) c h r o m a t o g r a p h y in a I X 16 cut gtass columiI. A linear gradient ( 2 4 0 ml) from 0 to 0.5 M KCi was applied to the c o l u m n and 2-ml fractions were collected in a Gilson microiYactionator. Protein was estimate d by measurit~g the absorbance o f the effluent at 280 rim.
3. Results Direct measurement o f dehydrogenase activity in high-speed supernatant liquids revealed ~outinely very 1! I
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weak reduction o f pyridin e nucleotides w i t h high blank values. Acrylamide gel 91ectrophorcsis however, revealed at least 6 distinct bands o f e n z y m e activity. (fig. 1). Bands 1 to 3 were Visible whe n the m e d i u m contained D-gi.ucose and NADP~=. The pair D-xylmie-- NADP + Was also active although at a much lower rate. B a n d 4 appeared w i t h glucose as h y d r .ogen dot3o r with either NAD* or NADP + as acceptor.:Band 5 w a s observed with th.e pair D - a r a b i n o s e - N A D p + o n l y when high a m o u n t s o f extract were electrophoresed. Band 6 was always present as a rapidly and. heavily .~ stained band when D.arabin0se ( o r L-fuc0se) and N A D + were present in the reaction mixture. ~ .Isolation and partial purification o f the e n z y m e corresponding to band 6 was accomplished b y DEAEcellulose column c h r o m a t o g r a p h y (fig. 2). A thin, : : syinmetricaJ peak eluted from the c o l u m n at about 0.13 M KCi. A t t e m p t s to com:entrate .the ¢.nzyme:
activityby ammonium su/fate,precipitation,or:fr_eezeT~. 112
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drying ~esulted in the almost c o m p l e t e i r e s . o f the activity. Thu.s, w i t h m t t further pudfi.~, tton the fractiom were U ~ d for e n z y m e characterization., .. Substrate spec~u:ity'studies are d~own m table ! using N A D ÷ as hydro~gen accept o r i n a s m u c ~ as NADI" was c o m p ~ t e l y i,aactive. D-Arablnose is b y far the best txtbst~te, a l t h o u g h L ~ f u c o ~ and D-n'bose w e r e also a t ~ c k e d b u t to a much. lesser>ex, teni.~ The.effect o f D-.arab inose c o n c e n t r a t i o n on the velocity o f NAD.+ reduction and viceversa i s shown in fig. 3. Classical M i c h a e l i s - M e n t e n kinetics were obtained. T h e calculated K m values were 6.7 X 10 -4 M for D-azabinose and 2.96 X 10 - 4 M for N A D +. We Imve n o t p e r f o r m e d detailed analysis o f the reaction products but since t h e a d d i t i o n ofhydroxYlamine to lfle reap tion. mixture at pH 6.5 resulted m hydroxamate p r o d u c t i o n w e . p r e ~ t m e that an arabonolactone h the immediate p r o d u c t o f the o x i d a t i o n reaction. % . .
•
4.
Discussion.i '..'
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Thts report adds several e n z y m e s to the grow~g, list o f dehydxogehases that c a t a l y z e oxidation-reduction reactions o.f f r e e . s u g a r . T h e e n z y m e s fr.qm N ~ are present in very small a m o u n t s . F o r instance, hex0.
--
.
..
.
FEBS. L w I ' r E I ~
Febxuary 1 9 7 3
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Effluenl v ~ u m e . n d for D - ~ . _ i n o ~ . d e h y d r o g e n a s e activity (o----o--o1 and absorbauoe at 2 8 0 n m ( I. The arrow indicates the beginning o f the KC! gmdk'nt (-- - --). The heavy h o r i z o n t a l line u r d e r the peak o f e n z y m e acl/v/ty Indicates the fraczions pooled for s u b s e q u e n t studies.
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plot,. ii3
Volume 30. number 1
kinase and glucose 6-phosphate dehydrogenases are at least one thousand times more active than D-arabinose dehydrogenase. Thus, the pathways in which these delq, drogenases are involved must..bo,..of.nlinor~ ~'~an7...... :tit ative~poi~t~fi~ei~iio::iile Celli.ii!a:t::ieast£ / o m i ! ~ ::p6h~t~ o f:view 0~: energy pmdu¢tiQniHowe~t.:the:p~sSibiiity: of:their being:!inVolvedin bios~/nthetiepathwiiys llas :" n0tbeen':ruled :o~it:(see:fo~ iristanc~e::i]8]:): ( :.II: : our stt:dieSiwith D.arabinosedehydrogenasc from N. crassa reVeaL striking d i f f e r e n c e s w i t h o t h e r . D.arabinoseldehydr0genases sO far:: reported: For instance:, the enzyme:fromrs~aomo~ [6] .is able to utilize both NAD*.-and NADP+.; Ais0, the JVeurospora dehydrogenase .is different from the.pig enzyme whose preferred substrata is L-fucose [ 12]. Finally, it is also different from the D-arabinose dehydrogenase from rat liver which is active only with NADP + as hydrogen acceptor [ 1 I, 15]. Preliminary experiments on colonial mutants of 2"Vettrospora ha:,e shown interesting quantitativo differences in the amount of arabinose d e h y d r o g e n a ~ and will be soon reported.
Acknowledgements This work was supported by grants from the Comisi6n de Energi'a Nuclear, Comisi6n Investigaci6n Cientffica, Facultad de Medicina, y Comisi6n Central de lnvestigaci6n Cientt~ca, Universidad de Chile. We thank Miss Jasna Radojkovi~ and M,ss Maria Riqttelme I'or their help in the early phases of this study.
114
'
FEBS LETTERS
February 1973
References i
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