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WAITE INSTIT'UTE tt,8.96 LIBRARY

IN THB ENZYMBS OF NITRATE ASSII',IILATION

SCLEROT IN T A SCLEROT I ORU I'1

by ì4askuntjir Abdul Rachim, B'Sc" Tr'

A thesis submitted in fulfilment of the'requirements for the degree of

Nlaster of Agricultural Science

Departmenr of Agricultural Biochemistry Waite Agricultural Research Institute The UniversitY of Adelaide'

FebruarY, 1986

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PREFACE

Part of the work described in this thesis has been pre- sented at the Australian þiochemical Society Conference (Canberra, 1985) and published in the following journals:

I Some properties of glutamine synthetase and glutamate synthase from ScTerotinia scl-er otiorun. M.A. Rachirn and D.J.D. Nicholas (f985) Proc. Aust. Biochen. Soc. !, 2I

2. Glutamine synthetase and glutamate synthase from Sc-lerotinia sclerotiorun. M.A. Rachim and D.J.D. Nicholas (1985) Phytochemistry 4, 254I-2548

3 Some properties of nitrate reductase from Sclerotinia scTerotiorun. M.A. Rachim and D.J.D. Nicholas (1986) Phytochenistry ë- (i" Press) 1L

ACKNO\^II-EDGEMENTS

f wish to express my deepest and sincerest thanks to my supervisor, Prof. D.J"D. Nicholas, chairman of the Department of Agricultural.Bio- The University of chemistry , lrlai-te Agricultural Research Institute ' and constructive criticism Adelaide for his constant encouragement ' guidance throughout the progress of the present investigation and in the preparation of manuscriPt.

I would also like to thank Mr. D. Hein for his assistance with the 15N Mr. B.A. Palk for preparing the photographic prints' "*p"riments, others who l"Irs. 1"1. Brock for the skrlful typing of the thesis and to a1I

helped me from time to time.

I am grateful to my wife Erna for her patience and unfailing

encouragenent dur-'ng the course of this investigation.

The postgraduate scholarship frorn International Development Programme of Australian Universities and Colleges is gratefully acknowledged ' a1a

DECLARATION

I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any Univerqity. To the best of my knowledge and belief no material described herein has been previously published or written by another person except when due reference is made i-n Ëhe text.

If accepted for the award of a M.Ag.Sc. degree, this thesis will be available for loan and photocopying.

}{.A. RACHIM l_v

ABBRBVIATIONS

The abbreviations for chemicals and symbols in general follow either the tentative rules of IUPAC-IUB Commission on Biochemical Nomenclarure (Biochen. J. (1966) 101: 1-7) or the Instruction to Authors for the Phytochernistry (Phytochemistry (1983) 22'- I-7).

CHBI.,I]CALS

ADP Adenosine 5t-diphosphate t AMP Adenosine 5 -monophosPhate | ATP Adenosine 5 -triphosphate BSA Bovine serum albumin BV Benzyl viologen BVH Benzyi viologen (reduced) CDP CyEidíne 5'-diphosphace | CMP Cytidine 5 -rnonophosphate CMP Cytidine 5' -triphosphate CyL b557 Cytochrome b55l DCPIP 2, 6-DichlorophenolindoPhenol DEAE-ce11u1ose Diethylaminoethyl ce11ulose DTT Dithiothrei-tol EDTA Ethylenediamine tetraacetic acid FAD Flavin adenine dinucleotide FADH2 Flavin adenine dinucleoticle (reduced) FMN Flavin mononucleotide Þ-MNH2 Flavin mononucleotide (reduced) GDP Guanosine 5t-diphosphate GMP Guanosine 5r-monoPhosPhate t GTP Guanosine 5 -triphosphate IDP Inosine 5r-diphosphate | IMP Inosine 5 -monophosPhate t ITP- Tnosine 5 -triphosphate MSX L-methionine-DL-Su1 Phoximine MV Methyl viologen 14VH Methyl viologen (reduced) NADH Nicotinamide adenj-ne dinucleotide (reduced) NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized ) NADPH Nicotinamide adenine dinucleotide phospiiate (reduced ) NEM N-ethylmaleimid e p-CMB p-chloromercuriben zoaLe SDS Sodium dodecyl sulphate -SH Sulphydryl group SVD Snake venom phosPhodiesterase TCA Trichloroacetic acid Tris Tris (hydroxymethyl) aminomethane | TIDP Uridine 5 -diphosphate | IJMP Uridine 5 -monophosphate t UTP Uridine 5 -triphosphate v

Svmbols and Units

A absorbance OC degree Celcius (centigrade) cm centimeter g gram o Ò unit of gravitational field hr hour(s) kDa kilo dalton(s) Ki inhibitor constant Michaelis-Menten constant K^lil L licre M molar mA milliampere mg milligram min minute(s) ml millilitre mm millimeter mlul millimolar mmo 1e millimo1e(s) l4l^l molec.¡lar weight ug microgram u1 microlitre urn micrometer u¡4 micromolar ( umo 1e micromole s ) N normal nm nanometer nmole nanomole ( s ) 7" percent inorganic phosphate Pa p S l- pound per square inch U V. ultra violet v volume vl weight va

TABLE OF CONTENTS

Ig. lto:_

PREFACE i

ACKNO\^TLEDGEMBNTS ii

DBCLARATION iii

ABBREVIATIONS iv vi TABLE OF CONTENTS

LIST OF TABLES ix

L]ST OF þ-IGURES xi

SUI'{l'{ARY xiv

1. INTRODUCTION 1

1.1 The biology of ScTerotinia scTerotiorun 1 L.2 Nitrate assimilation in microorganisms 3 ,.I rl 'iÌ T.2.I Nitrate reductase 4 ¡ L.2.2 Nitrite reductase 8 I.2.3 I'letabolism of ammonia into amino acids 11

r.2.3.r Glutamine synthetase L2 r.2.3.2 Glutarnate synthase L6

1.3 Aims of the study 18

2. MATERIALS AND METHODS 19

2.I Culturing Sclerotinia scTerotiorun 19

2.I.L Grov¿th conditions I9 2.L.2 Harvesting 19

2.2 Enzyme methods 20

2.2.I Nitrate reductase assay 20

I I 2.2.L .I Colorimet::ic 20 I 2.2.I.2 SPectroPhotometric 2I

2.2.2 Nitrite reductase and hydroxylamine 2L reductase assays 2.2.2.L Colorimetric 2l t 2.2.2.2 SPectroPhotometric 23 vl1

Paee No: 2.2"3 Glutamine sYnthetase assay 23

tl 2 "2.3 .r Transferase reaction 23 2.2.3.2 Biosypthetlc reaction 24 2.2.4 Glutamate synthase assay 25 2.2.5 Determination of Michaelis-Ilenten 25 constant (Kr) 2.2.6 Determinatiön of inhibitor constant (K1 ) 25 2.2.7 Determination of molecular weight 25 2.2.7 .I Gel f iltration 25 2.2.7.2 Electrophoresis 26 2.2.8 Separation and identification of flavin 27 from nitrate reductase 2.2.9 Adenylylation and deadenylylation of 27 glutamine synthetase

2.3 General techniques 29 15N 2.3.r Incorporation of into washed felts 29 2.3.2 Preparations of columns 29

2.3.2.1 Matrex Ge1 Blue A 30 2.3.2.2 Blue Sepharose CL-68 30 1 2.3.2.3 Sepharose 68 30 ef 2.3.2.4 DEAE-cellulose 31 'iù 2,4 Other determinations 31 2.4.r Nitrite 31 2.4.2 Ammonia 32 2.4.3 Protein 32 2.5 Biochemicals, chemicals and other materials 33 2.5.r Biochemicals and chernicals 33 2.5.2 Other materials 34

3. RESULTS 35

3.1 Nitrate reductase 35

3.1.1 Purification 35 3 "r.2 Properties 36 3 | .2.r Molecular weight 36 3 I.2.2 Effect of pH 36 I 3 r.2.3 Electron donors 36 3 r.2.4 K, values for substrate 4r reductant and cofacEor ' 3.r ,2.5 CharacterízaLion of flavin 4I isolated from the Purified enzyme t 3.1..3 Inhibitor studies l+7 vl_al

Paee No:

3.2 Nitrite reductase 55 3.2.r Purification 55 3.2.2 Properties 56 3.2.2,I Effect of ptl and time of incubation 56 3.2.2.2 Electron donors 60 3.2.2.3 Requirement for flavin 60 3.2.2,4 K, values for substrate ' reductant 60 and cofactor 3.2.2.5 Stoichiometries for NADPH, nitrite 66 and anmonia 3.2.3 Inhibitor studies 66 3.2.4 Inactivation of nitrite and hydroxylamine 70 reductases by NADPH in the Presence of FAD

3.3 Incorporation of [15tq]-fuUe11ed (NH4)2504 into cell- 70 nitrogen 3.4 Glutamine sYnthetase 75 3.4.1 Purification 75 3,4.2 Properties 77 3.4.2.r Molecular wei-ght 77 3.4.2.2 Effect of pH and incubation time 77 3.4,2.3 Divalent cation requirenent 77 3.4 .2.4 Nucleotide specificitY 82 3.4,2.5 K, values for substrate 82 3.4.3 Inhibitor studies B4 3.4.4 Adenylylation/deadenylylation 89 3.5 Glutamate sYnthase 95 9s 3.5. 1 Purification 3.5.2 Properties 99 3.5.2.r Molecular weight 99 3.5.2.2 Effect of pH 99 3.5.2.3 Substrate requirement 99 3.5.2.4 K, values for substrates and NADPII 99 3.5.3 Inhibitor studies 103

t12 4. DISCUSSION 4.r Nitrate reductase tr2 4,2 Nitrite reductase TT7 4.3 Pathway of ammonia assimilation L22

I 4.3.1 Glutamine synthetase t23 4.3.2 Glutamate synthase r27

5. BIBLIOGRAPHY 131 I l-x

LIST OF TABLES

Table Pase No:

1 Purification of nitrate reductase

2 Electron donors for the purified nitrate reductase activitY

3 Effects of metal inhibítors on the MVlI-dependent nitrate reductase activitY

4 Effects of inhibitors on NADPH-dependent nitrate reductase activitY

5 Effects of inhibitors of sulphydryl-groups and flavin respectively on MVH-nitrate reductase activity

6 Purification of nitrite reductase

7 ¿ffects of various electron donors on nitriEe reductase activitY

B Effects of flavins on NAD(P)H-nitrite reductase and its associated diaphorase activities

9 Stoichiometries for NADPH, NOt and NH3 for nitrite reductase

10 Effects of metal binding agents on NADPH- dependent nitrite reductase

11 Effects of inhibitors of sulphydryl-groups and flavin on NADPH-nitrite reductase I2 Effects of preincubation of nitrite reductase with reductant, cofactor andfor substrate on its activity

(MSX) 13 Effects of L-methionine-Dl-sulphoximin e - and azaserine on the incorporation of r5N- label1ed (NH4)2504 into washed cel1s

t4 Purification of glutarnine synthetase

15 Transferase and biosynthetic activities with various divalent cations

16 Effects of various nucleotides on transferase and biosynthetic activities t7 Effects of various concentration of L-methionine- Dl-sulphoximine on transferase activity

18 Effects of various amino acids on glutamine synthetase activi-LY x

: i ì

I Pase No: Table I 91 19 Bffects of organic acids on glutamine synthetase activitY

L on 20 Effects of snake venon phosphodiesterase 96 glutamine sYnthetase activitY 2t Purification of glutamate synthase 98

22 Substrates and NADPH requirements for ro2 glutamate sYnthase activitY

23 Effects of amino acids on glutamate synthase 107 activitY

24 Effects of various metabolites on glutamate 108 synthase activitY

25 Effects of various concentrations of azaserine 109 on glutamate synthase activity

26 Effects of various inhibitors on glutanate 110 synthase activitY xl-

LIST OF F]GURES

Figure Pase No: 38 1 A. llolecular weight determination of nitrate reductase bY ge1 filtration B. Estimation of subunit molecular weight of the purified enzyrne by SDS-polyacrylamide ge1 electroPhoresis

39 2 Effect of pH on nitrate reductase activity 42 3 Double reciprocal plots of the effects of various nitrate concentrations on NADPH- and MVH-clependent nitrate reductase activities

4 Double reciprocal plots of the effects of 43 various concentrations of nitrate on FMNH2- and FADH2-linked enzyme activlties

5 Double reciprocal plots of the effects of 44 varying NADPH and FAD conceritrations respectively on NADPH-dependent nitrate reductase activitY

6 A double reciprocal plot of the effects of 45 various concentrations of reduced methyl- viologen on nitrate reductase activity

7 Separat-ion of the flavin component of the 46 purified nitrate reductase b1' paper chroma- tography I Inhibi-tory effects of chlorate and bromate 51 on l4VH-nitrate reductase activity

9 A Dixon plot of the inhibiiory effects of 52 various concentrations of chlorate on the MVH- nitrate reductase activitY

10 A Dixon plot of the inhibitory effects of 53 various concentrations of bromate on the MVH- niErate reductase activitY

11 Effects of various concentrations of nitrite 54 on NADPH-nitrate reductase activity

L2 Effects of pH on nitrite reductase and hydro- 58 xylamine reductase activities

13 Effects of incubation times on nitrite reduction 59

T4 Effects of various concentrations of substrates 63 on NADPH-nitrite reductase and NADPH-hydroxylamine reductase activities XII

Figure Page No:

15 Double reciprocal plots of the effects of 64 various concentrations of NADPH on NADPH- dependenL nitrite reductase and NADPII- dependent hydroxylamine reductase activitj-es

16 Double reciprocal plots of the effects of 65 various concentrations of FAD on NADPH- dependent nitrite reductase and NADPtI-hydroxylamine reductase activities t7 Effects of various concentrations of cyanide 7l on NADPH-dependent nitrite reductase activj-ty

18 Effects of various concentrations of sulphite 72 on NADPH-dependent nitrite reduction

19 A. Molecular weight determination of gluEamine 78 synthetase by gel filtration B. Estimation of subunit rnolecular weìght of the purified enzyme by SDS-polyacrylamide gel electrophoresis

20 Effects of pH on glutamine synthetase activity 79

2l Effects of incubation times on g,lutarnine BO synthetase activity

22 EffecEs of various concentrations of substrates B5 on glutamine synthetase- transferase activity

23 Effects of various concentrations of substrates 86 on glutamine synthetase-biosynthetic activity

24 Effects of various concentrations of ADP and 87 ATP on glutamine synthetase activity

25 Inhibitory effects of glutamate and NH4C1 on 92 glutamine synthetase-transferase activity

26 A Dixon plot of the effects of various concen- 93 trations of glutarnate on glutamine synthetase- transferase activity

27 A Dixon plot of the effects of various concen- 94 trations of ammonium chloride on glutamine synthetase-transferase activity

28 A. Molecular wei-ght determination of glutamate 100 synthase by gel filtration B. Estimation of subunit molecular weight of the purified enzyme by SDS-polyacrylanide gel electrophoresis xl-11

Paee No: Figure 101 29 Effects of pH on glutamate synthase âctivity

30 Effects of various concentrations of glutamine 104 on glutamate sYnthase activitY

31 Effects of various concenLrations of a-keto- 105 glutarate on glutamate synthase activity on 32 Effects of various concentrations of NADPH 106 glutamate sYnthase activitY xav

SUI'IMARY

biochemical l. This thesis embodies results of an invesLigation on some in a plant- aspects of enzymes involved in nitrate assimilarion are pathogenic fungus,scTerotinia scTerotiorum' The enzymes nitrate reductase, nitrite reductase, glutamine synthetase and glutamate sYnthase.

2.Nitratereductase,purifiedll8-fold'hadamolecularweightof I23 and 107 kDa' 210 kDa and was composed of.2 dissimilar subunitsof also In addition to using NADPII as an electron donor, the enzyme utilizei reduced viologen dyes and reduced flavin nucleotides as reductants.

however, exogenous FAD was 3. FAD was isolaÈed from the purified enzyrne; reductase in required for rnaxj-mal activity of NADPH-dependent nitrate

vitro. FMN di-d not substitute for FAD' agents and by 4. The enzyme activity was inhibited by metal chelating flavinandsulphydrylgroupinlribitors.Ãzidemarkedlyrestricted by p-cMB, NEùl both NADPH- and l'lvH-dependent reactions, but inhibition

and amytal was more marked when NADPH was the reductant' nitrate 5. Nitrite inhibited nii.rate competitively in NADPl{-dependent reduction in vitro; however, it is unlikely to inhibiE nitrate ttre K1 for reductase activity under physiological conditions because nitrite was three-fold greater than the K, lor nitrate '

6. Nitrite reductase, the second enzyme in the nitrate assimilation flavirt pathway utilized NAD(P)H, reduced viologen dyes and reduced electron nucleotides as electron donors. NADPH was the most effective donor. Maximal activi-ty of Ehe NADPH-dependent nitrite reduclase XV

hras achieved by adding FAD to the assay mixture; FMN however' I^/as less effective.

7. Associated with the nitrite reductase enzyme v/as a hydroxylamj-ne reductase activity as well as diaphorase type activities utilizing either ferricyanide, DCPIP or cytochrome c as an electron acceptor.

B. The product of both nitrite and l-rydroxylamine reductases was arnmonia but hydroxylamine \^/as not an intermediate product of nitrite reductase.

The stoichiometry of NADPH oxidized to nitrite ut'ilized and ammonla

produced in NADPH-dependent nitrite reduction was 3:1:1 and the ratio

of NADPH oxidj-zed t9 ammonia forned in the l-rydroxylamine reductase- mediated reaction was 1:1. g. In the presence of FAD, nitrite reductase rvas j-nactivat-ed by preincu-

bation with NADPH, but NADP+ was without effect. This inactivation

was offset by the substrates (nitlite and hydroxylamine).

10. Nitrite reductase was sensitive to metal binding agents as well as flavin- and s.rlphydryl group-inihibitors. The inhibition by p-Cl4B and mepacrine respectively, were reversed by adding cysteine and FAD. l5llH4* 11. I^lashed f elts readily incorporatea irrto cell-nitrogen but this effect was inhibited by both MSX and azaserine, inhibitors of glutarnine synthetase and glutamate synthase respectively. Since glutamate

dehydrogenase was not detected in cel1-free preparations, this is further evidence that the glutarnine synthetase/glutamate synthase pathway is the main route for the assimilation of ammonia into amino acids.

12. Glutamine synthetase, purified by ion exchange and affinity chroma- tography, had a molecular weight of 490 kDa. The enzyme was composed of B identical subunjts of 60 kDa. The transferase activity of the xvl"

whereas the bio- enzyme required Mn2* und ADP for maximal activity synthetic activity required Mg2+ and ATP' amino acids 13. The enzyme was regulated by feedback inhibition involving mechanism' and organic acids and by a adenylylation/¿eadenylylation competit- The transferase activity of the eÛzyme was also inhibited ively by the substrates of the biosynthetic reaction, namely glutamate

and NH4CI, with respect to glutamine '

was composed 14. Glutamate synthase had a molecular weight of 22O kDa and of 4 subunits of 53.7 kDa. The enzyme had a specific requirement donor, amino for NADPH, cr-ketoglutarate and glutamine as the electron acceptor and amino donor, respectively' including l-5. Glutamate synthase was regulated by feedback inhibitors was rnarkedly amino acids, organic acids and nucl,eotides. The enzyme inhibited by p-cllB (this effect was reversed by cysteine), ' O-phenanthroline, o,o'-dipyridyl and azaserine'