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A Genetic Strategy to Reduce Sulfite Reductase Activity In TY i >t'tt I, >t f rrlo0 A genetic strategy to reduce sulfite reductase activity in S acch ør o my ces cer evisiøe. by CATHERTNE M. SUTHERLAND BSc.(Hons) (Adel.) A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Agricultural and Natural Resources Sciences Department of Plant Science The University of Adelaide May 2000 TABLE OF CONTENTS THESIS SUMMARY..... 1 DECLARATION.. .4 ACKNOWLEDGEMENTS.... .5 CHAPTER ONE INTRODUCTION. .6 1.0 INTRODUCTION ........... .6 1 2.0 SULFUR ASSIMILATION............ 3.0 BIOSYNTHESIS OF CYSTEINE AND METHIONINE.."". .8 4.0 GENETICS OF SULFATE ASSIMILATION ..........' 10 5.0 REGI.'LATION OF TIIE SULFATE ASSMILATION PATTIWAY. " . " . .. .. t2 5.1 THB nol-e oF S-ADENosYLMETHIoNINE....".....'.. t2 5.2 TRnNscRI¡T IoNAL coNTRoLoF METGENES .......... 13 5.3 Tne nole oFTHEMET4 PRoTEIN T4 5.4 THe RoleorïrsMer30 PoLYPEPTIDE t7 18 5.5 Posr-TRANSCRIPTIoNAL coNTRoL.. .. 6.0 SULFITE REDUCTASE............... 6. 1 S¡ccn¡nouvcøs cEREVISIAE SULFITE REDUcTASE. ' 6.2 EScHERICHIA co¿I SULFITE, REDUcTASE.. 7.0 GENETIC ENGINEERING OF YEAST TO REDUCE H2S PRODUCTION"" 8.0 CONCLUSIONS............. CHAPTER TWO CHAPTER THREE 58 3.01 INTRODUCTION........... .58 3.02 RESULTS .61 3.O2.1 MoDELLINc rHE 3-DIMENSIoNAL sTRUCTURE oF MET10... .61 3,O2.2 SELESTION OF AMINO ACIDS TO 4LT8R....,.... .61 .N¡ECNPRN"TER' 3.02.3 SITE OtnECTr,D MT-TTAGENESIS - THE METHOD.. .64 3.O2.4 Slre olReCTeD MUTAGENESIS - OLIGONUCLEOTIDE EXTENSIoN TECHNIQUE.. .65 3.02.5.1 Viability of dominant negative mutants in the absence of methionine.. 3.02.5.2 Sulfite reductase measurements 3.02.5.3 Determination of sulfide accumulation by growth on indicator medium...... 3.02.5.4 Determination of sulfite accumulation 3.03 DISCUSSTON .............. 3.03,I SITE-DIRECTED MUTAGENESIS.,.........,.... 3.03.2 TrrE EFFECT OF AMINO ÀCID SUBSTITI.ITION W MET1OP OT'I SUITITE REDUCTASE 69 ACTIVITY IN S. CEREYISIÁ890844 CHAPTER FOUR IDENTIFICATION OF PROTEINS THÄT INTERACT WITH MET1OP 72 .72 4,01 INTRODUCTION......... .74 4.02 RESULTS... .74 4.02.I EXPRESSION OF MET1OP TN E. COLI.... .74 4.02.2 PUNMIC¡,TION OF DENATURED AND NATIVE METIOP'..... .75 4,02.3 DETESTION O¡ MBTIOP BY T}IE AI.ì"TI-METIOP ANTIBODY' .76 4.02.4 IMMUNoPREcIPITATIoN.. 'BAIT' ........ .76 4.02.5 Scnee¡¡rNC ¡, yensr CDNA l-nnnRy wmt MerlOP AS THE '... DETECTED 4.02.6 A DTRECT INTERACTION BETWEEN MET1OP NNO MET5P IS ONLY 77 IN THE ABSENCE OF METHIONINE ,,78 4.03 DISCUSSION............... 4.03.1.1 Protein modification ..81 4.03.1.2 Interaction of the a and p subunits via siroheme """"""""""" ..82 4.03.2 MET5P AND cELL wALL BIoSYNTI{ESIS .......... " ..83 4.03.3 Sulrrre roxIcITY..... ..84 4.04 coNcLUSroNS ............. ..85 CHAPTER FIVE EXPRESSION OF METLO CONSTRUCTS IN AMET(O STRAIN OF S. CEREVISIAE 87 5.01 rNTRODUCTION......... .""""""87 5.02 RESULTS ..""""""88 p SULFITE R¡OUCTRSE. .91 5 .02.4 THE ALTERED Cr, SUBLNTTS STrLL BrND THE SUnUln oF 5.02.7 SuLFnEREDUcrASEASsAY......'.. """'93 5.03 DISCUSSrON............... '..""""93 5.03.1 GlyCrNe TO ALANINE SUBSTITUTION WITHIN THE PREDICTeo NADPH BTNDTNG MOTIF......... """""""""""96 5.03.2 SERINE TO VALINE SUBSTITLTTION IN THE PREDICTED FAD ¡tNotNC Stre""""'97 5.03.3 SERINE TO LYSINE SUBSTITUTION IN THE PREDICTED NADPH BINDING SIr¡'" ' ' '98 5.O3.4 LYSINE To SERINE SUBSTITUTION IN THE PREDICTED NADPH BINDING slre " " '98 5.03.5 SugsrItutloN OF CYSTEINE wlTH ALANINE IN THE PREDICTED NADPH BINDING SITE,....,.,.... 99 CHAPTER SIX GENERAL DISCUSSION..... 104 APPENDIX ONE .10 8 APPENDIX TWO. .1 1.6 REFERENCES...... 125 THBSIS SUMMARY A genetic strategy to reduce sulfite reductase activity in S acc h ar omyces cerevisiøe. The production of hydrogen sulfide by Saccharomyces cerevisiae during wrne fermentation has long been a problem for wine makers as HzS has a low odour threshold. The problem occurs when yeast attempt to make the sulfur containing amino acids methionine and cysteine via the sulfate assimilation pathway, in a low nitrogen grape juice. In the presence of nitrogen, sulfide produced by the enzqe sulfite reductase combines with nitrogenous precursors to form methionine. If the nitrogenous precursors are not available the sulfide diffuses from the cell as hydrogen sulfide. This study was undertaken to derive a strategy to reduce the potential of ,S. cerevisiae to produce hydrogen sulfide under oenological conditions by altering the levels of active sulfite reductase in the cell. To achieve this, the study also looked at the structure/function relationships of sulfite reductase. A reduction in the levels of active sulfite reductase rather than the elimination of the enzyme was necessary, as methionine and cysteine are essential amino acids and yeast must be able to synthesise these if they are not available in the growth medium. The dominant negative strategy was chosen to accomplish this. Sulfite reductase is a member of the flavoenzpe family of proteins and catalyses the six electron reduction of sulfite to sulfide. S. cerevisi¿¿ sulfite reductase is a heterotetramer with an cr2B2 structure. The a subunit, encoded by METI0, binds the cofactors NADPH and FAD, whilst the B subunit binds FMN and siroheme to form the active enzyme. The interaction between the sulfite reductase subunits was examined using the yeast two-hybrid system. Previous experiments had indicated that the B subunit of sulfite reductase was encoded by MET5, and results from this study provide further evidence for this I as an interaction between Met5p and Metlþ was seen in the two-hybrid system. However the interaction could only be detected in the absence of methionine. Models that may explain why Met5p and Metl0p were only seen to interact when cells were grown under conditions in which the methionine biosynthetic pathway is on are proposed. The homology of sulfite reductase to other members of the flavoenzyme family enabled the three dimensional structure of the cofactor binding region to be modelled. Amino acids in the predicted NADPH and FAD binding sites were substituted to prevent cofactor binding, whilst still altowing the cr and p subunits to bind to each other, and the altered Metl0p over-expressed from a plasmid in a haploid s. cerevisiae strain containing the wild-tlpe Metl0 protein. The dominant negative effect in these cells of the amino acid substitutions within Metl0p on the activity of sulfite reductase was determined with a sulfite reductase et:zqe assay and a sulfite accumulation ¿rssay. No difference could be detected between strains of S. cerevisiae encoding altered Metl0p and a wild- tlpe S. cerevisiae, therefore further experiments were undertaken to find the reason for this. To ensure the amino acid substitutions had not altered the folding properties of Met10p such that it could no longer bind to the p subunit, subunit binding was tested. Yeast two-hybrid experiments showed that the altered cr subunits could bind and therefore sequester the sulfite reductase p subunit. A strain of S. cerevisiøe which had MET|0 deleted and appropriate auxotrophic markers was constructed and the effect of the amino acid substitutions on sulfite reductase activity estimated in this strain by sulfite accumulation assays. It was found that all of the mutants retained some activity, although three of the amino acid substitutions affected the activity of sulfite reductase to the extent that when these altered cr subunits were expressed in the Lmetl} strain, growth in the absence of methionine was not supported. 2 The amino acid substitutions introduced into Metl0p were all shown to reduce activity of sulfite reductase and would therefore be expected to produce a dominant negative phenotype when overexpressed in S. cerevisiae. The most likely explanation that a phenotlpe was not observed is that the level of expression of the metl| genes was not high enough when driven by the MET3 promoter. Based on the results presented it would appear that the dominant negative strategy is feasible to reduce the production of HzS in yeast, however increased expression of the altered subunit is required. The subsitution of selected amino acids within predicted co-factor binding sites in Metl0p, and the determination of their effect on sulfite reductase activity has provided some insights into the structure/function relationships within this enzyme. The observation of an interaction between Metl0p and Met5p in the yeast two- hybrid system provides further evidence to support the function of Met5p as the sulfite reductase B subunit. Models are proposed to explain why the interaction could only be observed in the absence of methionine. J DECLARATION This work contains no material which has been accepted for the award of any other degree ol diploma in any univelsity or other tertiary institution and, to the best of my knowledge, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Lrbrary, being available for loan and photocopying. Catherine Sutherland April2000 ACKNOWLEDGEMENTS Many people need to be thanked for helping and supporting me throughout my PhD, a-nd I would like to apologise to those who are omitted, but you know who are. Firstly, I would like to thank my supervisors, Professor Peter Langridge, Dr Paul Henschke, and Miguel de Ba:ros Lopes for their guidance throughout the project and the preparation of this thesis. I would especially like to thank Miguel who has been my biggest critic and a great'sounding board' for ideas - I am learning to ask the questions to find the answers. I also want to thank Dr Ute Baumann for many useful discussions both in the lab and over coffee, about my project and science in general. Thankyou to Jørgen Hansen for 'MET chats' via email - it was always quite helpfui.
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