Insights into the catalytic mechanisms of a 3-ketosteroid Δ1-dehydrogenase and a β- xylosidase obtained from 3D structures Rohman, Ali

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1 University of Groningen Insights into the catalytic mechanisms of a 3-ketosteroid Δ1-dehydrogenase and a β- xylosidase obtained from 3D structures Rohman, Ali IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Rohman, A. (2016). Insights into the catalytic mechanisms of a 3-ketosteroid Δ1-dehydrogenase and a β- xylosidase obtained from 3D structures [Groningen: Rijksuniversiteit Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Insights into the catalytic mechanisms of a 3-ketosteroid Δ 1 -dehydrogenase and a β-xylosidase obtained from 3D structures Ali Rohman

3 PrintedbyIpskampDrukkers,Enschede. TheresearchpresentedinthisthesiswascarriedoutintheProteinCrystallographygroupat thegroningenbimolecularsciencesandbiotechnologyinstitute,facultyofmathematicsand NaturalSciences,UniversityofGroningen,theNetherlands. Frontcover: The active site of the 3Iketosteroid Δ 1 Idehydrogenase isoenzyme 1 from Rhodococcus( erythropolissq1withboundreactionproduct1,4iandrostadienei3,17idione. Backcover: Photographs of crystals of the 3Iketosteroid Δ 1 Idehydrogenase isoenzyme 1 from Rhodococcus(erythropolis SQ1 (top) and the βixylosidase from Geobacillus(thermoleovorans ITI08(bottom)andexamplesoftheirXIraydiffractionimage. ISBN:978I90I367I9315I5(printversion) ISBN:978I90I367I9314I8(electronicversion)

4 Insights into the catalytic mechanisms of a 3-ketosteroid Δ 1 -dehydrogenase and a β-xylosidase obtained from 3D structures Proefschrift ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op dinsdag 6 december 2016 om 9.00 uur door Ali Rohman geboren op 7 augustus 1968 te Pati, Indonesië

5 Promotor Prof. dr. B.W. Dijkstra Beoordelingscommissie Prof. dr. W.J.H. van Berkel Prof. dr. M.W. Fraaije Prof. dr. M.J.E.C. van der Maarel

6 For my parents: Hasan Asy ari and Siti Rohmah my family: Nunik, Adi, Shofy, and Tya and all my gurus

7 (

8 Preface and acknowledgements Seeing is understanding. In many instances, seeing is the best way to understand natural phenomena. X:ray crystallography is among the most powerful methodstovisualize3dstructuresofproteinsandnucleicacidsattheatomiclevel.in X:ray crystallography, a protein crystal is exposed to X:rays, which generates a specificx:rayscatteringpattern,calledadiffractionpattern.thediffractionpatternis thenusedtoreconstructthe3dstructureoftheproteinmolecule.inthisthesis,the crystallographicmethodisappliedtoelucidatethestructureofthe3:ketosteroidδ 1 : dehydrogenase isoenzyme 1 (Δ 1 :KSTD1) from Rhodococcus( erythropolis SQ1, an organism closely related to Mycobacterium, and the β:xylosidase from the thermophilic bacterium Geobacillus( thermoleovorans IT:08 (Xyl). 3:Ketosteroid Δ 1 : dehydrogenases (Δ 1 :KSTDs) are pharmaceutically important enzymes as they are appliedfortheproductionofδ 1 :3:ketosteroidaldrugsandhormones.Incombination withmutationalanalysis,thestructureofδ 1 :KSTD1clarifiesthecatalyticmechanism of Δ 1 :KSTDs. Furthermore, the Δ 1 :KSTD from Mycobacterium( tuberculosis was identifiedtoplayanimportantroleintheetiologyoftuberculosis.thus,thestructure ofr.(erythropolissq1δ 1 :KSTD1mayalsofacilitatethedesignofinhibitorsthatcould possibly be developed into new anti:tuberculosis drugs. Regarding the second subject, β:xylosidases are hemicellulolytic enzymes that are of biotechnological interest for saccharifying hemicellulosic biomass. However, these enzymes are commonly inhibited by monosaccharides produced during the saccharification process.byanalyzingthe3dstructuresofxylincomplexwithitsnaturalinhibitors, i.e.l:arabinoseandd:xylose,welearnedhowtheinhibitorsinhibittheactivityofthe enzymeandoffertentativesolutionstoovercometheinhibitionproblems.ihopethat we,andespeciallyme,couldtakesomeadvantagesfromthisthesis. Afterall,itisquiteemotionalformetowritetheseacknowledgements.Istilldo notbelievethatseveraldaysagoitypedthelastwordofmythesis.itwasareally hard work. It is true that I started this PhD study with an experience in protein crystallization,i.e.xyl(chapter5).however,thisexperiencewasfarfromsufficient. ThereweremanyobstaclesIhadtofaceduringthisstudy,includingfamilyproblems, financial limitations, lack of academic and lab work skills as well as language proficiency,etc.itwasalsoverysadformetospendmorethanoneyeartoworkon HsaC, a steroid:degrading enzyme from M.( tuberculosis, but it was ended by the appearanceofthestructureoftheenzymepublishedbyanothergroup.fortunately,i had many good people who helped me addressing those obstacles, eased my difficulties,andinspiredmetocompletemyphdstudy.ofcourse,iamveryhappyto 7

9 Preface and acknowledgements writethisforeword.itisoneofpleasantparts of my life. In this joyful moment I wouldliketoexpressmysinceregratitudetothosepeople. Firstly,IwishtoconveymysincerethanksandappreciationstoProf.Bauke,my kind supervisor. It was a great honor for me that he accepted me as his student. Furthermore, he assisted me arranging administrative documents required for my PhD study. Subsequently, he provided me great scientific supervisions, including helping me a lot in writing journal articles and this thesis. It was also fun to have potluckdinnerswithallmembersofthex:rayandelectronmicroscopygroupsinhis houseeveryendofdecembereachyear.inthisrespect,mythankisextendedtopaul Herruerasthehostofthedinner.Paulalsodidproof:readingofthe Samenvattingen conclusies chapter of this thesis, which was reviewed and translated to Dutch by Prof. Bauke. Overall, I do not think that my thanks to Prof. Bauke can be entirely describedbywordsorrepresentedbyfigures.however,itisimprintedinmyheart. I would like to also thank all (including the former) members of X:ray and ElectronMicroscopygroups.Theywerehelpfulandmademylifeinthelabeasier.My thanksgotoprof.egbert,andy:mark,jaap,andankefortheirhelpfuldiscussionsand suggestions,particularlyduringgroupmeetings.andy:markalsohelpedmerefining thestructureofδ 1 :KSTD1.ToPakCor,Tjaard,Henriette,andJohan,thankyouvery much for helping me solving the lab troubles. Furthermore, it was very nice to stay withthesex:ray and Electron Microscopy people(sorted alphabetically): Abhishek, Avinash,Dejan,Dmitry,Emilie,Eswar,Gjalt,Guntur,Harshwardhan,Hendrike,Ilona, Jelle, Jinfeng, Maarten, Marcel, Marco, Milena, Niels, Paul Tucker, Pramod, Roman, Satish, Sonja, and Kang Wangsa. Thank you all for discussions, friendliness, and havingcoffeebreakandlunchtogether.myspecialthankgoestoniels.amongmany things, he spent a lot of time to teach me Protein Crystallograpy 101, offered me to work on Δ 1 :KSTD1 (a project that was started by him), and introduce me to some sweet Dutchdrop.MysincerethanksarealsoforHildaandSandra,thededicated secretariesofx:raygroup,whohelpedmeinalladministrativethings. Thisthesiswillbenotcompletewithoutbeingreviewedbyareadingcommittee anddefendedinadefenseceremony.therefore,iwouldliketothankthemembersof thereadingcommittee,namelyprof.dr.w.j.h.vanberkel,prof.dr.m.w.fraaije,and Prof. dr. M.J.E.C. van der Maarel, for spending their valuable time to read the thesis manuscript and providing critical inputs. My sincere appreciations go to all the opponents of the defense for making their time available to discuss my thesis with me. Finally, to Niels and Fean, thank you a lot, guys, for your willingness to be the paranymphsinthedefense Furthermore, my deepest gratitudes are to my family. To my father, Hasan Asy ari,andmylatemother,sitirohmah,thankyouveryverymuchfortakingcareof mewheneverineed.idonotbelievetotheindonesianproverbstatingthatparents 8

10 Preface and acknowledgements love is as long asthe road. It is even longer and everlasting. To my brothers and sister:in:laws, Kang Dirman and Mba Mudah, Kang Kan and Mba Laila, Kang Hadak and Mba Is, and Kang Man and Mba Is, thanks a lot for supporting me and encouraging me to complete my study. To my wife, Nunik Emiwati, thank you very muchforeverything,especiallyyourpatienceintakingcareofourchildrennotonly when I was away in Groningen but also when I am at home. To my children, Adi, Shofy,and Tya, thanks for beinggoodchildrenandunderstandingyourfather.you arethereasonbehindthisstudy. Mybestwhishesareforallofmygurus.Myapologiesfornotabletomentionall their names here. However, in short, they brought me from a farming field to a biochemistrylab.dearibususmiati(formerlyfromsmanlasem),prof.ami(retired from Universitas Airlangga), Prof. Usman Sumo (University of Indonesia), and Prof. Bauke, please accept my sincere gratitudes. They influenced me a lot to love chemistry, organic chemistry, biochemistry and bioinformatics, and protein crystallography,respectively. IalsothankGroningen.Itisaverypleasantplacetolivein.Thecityisorganized verywell,asothercitiesinthenetherlands,andnotsobusy.thepeoplearealsovery nicetoastrangerslikeme.besides,itisveryeasytofindindonesian/asianfoodand ingredientsingroningen.itisevenpossibletofindtraditionalindonesianfoodsthat are no longer popular in Indonesia itself. Moreover, Groningen has quite a big Indonesian community(degromiest and PPIG), most of their members are students oftheuniversityofgroningen.iamthankfultoallmembersofdegromiestandppig for periodically arranging Indonesian gatherings,andthusproviding an Indonesian atmosphere in Groningen. To mention a few, they are (sorted alphabetically) Mas Adhi, Alia, Amel, Astri, Bintoro, Daniel, Dini, Teh Enci, Eryth, Faizah, Fean,Pak Gani, Insanu,PakIntan,TehIntan,Irfan,MasIzul,BliKadek,Lia(Atok),Muhsin,Muis,Kang Nandang, Neng, Teh Nisa, Nizar, Pandji, Mba Puri, Puti, Rachma, Mba Sista, Mba Sri, MbaTina,TehUyung,Wahono,andYusuf.KagemsesepuhdiGroningen,WakAsiyah sarimbit,pakwidsarimbit,pakhenk,danbucora,maturnuwunatassambutannya yang hangat kepada saya. I also would like to extend my thanks to Mas Donny and MbaNiekeforallowingmetooccupyaroomintheirhouseduringmy3:monthsstay ingroningen.myveryspecialthankstokangwangsa,guntur,iging,anddesti.they helpedmeinmanyrespects,includingroomarrangementformetoliveingroningen. KangWangsaandGunturalsohelpedmealotinthelab.ForinhabitantsofNieuwe Kerkhof 30A, E.H. du Marchie van Voorthuysen (the landlord), Pak Mustajab,and especiallyduoanakmudarobbyandiqbal,thankyouveryverymuchforbeinglikea familytome. 9

11 Preface and acknowledgements Moreover,Iwouldliketoexpressmyhighestappreciationtothebigfamilyof thedepartmentofchemistryandtheinstituteoftropicaldisease(itd),universitas Airlangga.Thankyouallverymuchfor,amongothers,shapingacomfortableworking environmentinourofficeandcheeringmeuptocompletemyphdstudy.particularly, my sincere thanks go to Prof. Ni Nyoman for introducing me to Prof. Bauke, and facilitating my study. It is a pleasure for me to work with her and I wish we could worksynergisticallyaftercompletingthisstudy.toibutjitjik,theformerheadofthe Department of Chemistry, you have my deepest gratitude for giving me the opportunity to join the Biochemistry Group at the Department. To Prof. Inge, the DirectorofITD,thankyouverymuchforallowingmetoworkinITD.IhopeIcan contribute optimally in the close future. For the sake of friendship, I thank a lot Bu Gondo Mastutik and Pak Ucok for chatting about life and continuing updating my thesiswriting. ThisstudyisapartofmytasksasalecturerintheDepartementofChemistry, FacultyofScienceandTechnology,UniversitasAirlangga.Iamhighlyobligedtothe Rector of Universitas Airlangga, the Dean of the Faculty of Science and Technology, andtheheadofthedepartmentchemistryforallowingmetopursuemyphdstudy, helping me to get a scholarship, and providing many other assistances. I am also indebtedtopeopleinbagiankependidikan,universitasairlangga,especiallybudiah, for arranging my scholarship and other administrative stuffs.thisstudyhad been virtually impossible without financial support. Therefore, I would like to acknowledgetheministryofresearch,technology,andhighereducation,republicof Indonesia,forprovidingascholarshiptomeforthisstudy. Ofcourse,therearestillmanymorepeople/institutionsIwouldliketothankfor theirvariouscontributionstomystudy.however,iamnotabletomentiononeby one. I do not forget them; instead, nature makes me forget (to remember them). Pleaseacceptmyapologies.Thus,Ithankthemverymucheveniftheirnamesarenot printedhere. 10

12 Table of contents Table of contents 11 1 Microbial 3-ketosteroid Δ1 -dehydrogenases: biochemistry and biotechnological applications Purification, crystallization, and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ 1 -dehydrogenase from Rhodococcus erythropolis SQ1 61 Crystal structure and site-directed mutagenesis of a 3- ketosteroid Δ 1 -dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism 73 β-xylosidases: structural diversity, catalytic mechanism, and inhibition by monosaccharides 93 Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT Crystal structure of a thermostable glycoside hydrolase family 43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08 in complex with arabinose and xylose 121 Summary and concluding remarks 139 Samenvatting en conclusies 147 References

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