Biosynthesis and function of biological pteridines [Elektronische Ressource] : structural studies on two molybdopterin containing aldehyde oxido-reductases, from Desulfovibrio desulfuricans ATCC 27774 and from Desulfovibrio gigas, and the GTP cyclohydrolase I on E. coli, responsible for the first step of the tetrahydropterin biosynthesis / Jorge Manuel Baeta Simões Rebelo

Rebelo

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 2 Mo

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Max-Planck Institut für Biochemie
Abteilung Strukturforschung




Biosynthesis and function of biological pteridines

Structural studies on two molybdopterin containing Aldehyde oxido-reductases, from
Desulfovibrio desulfuricans ATCC 27774 and from Desulfovibrio gigas, and the GTP
cyclohydrolase I on E. coli, responsible for the first step of the tetrahydropterin
biosynthesis.



Jorge Manuel Baeta Simões Rebelo

Vollständiger Abdruck der von der Fakultät für Chemie der
Technische Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Dr. Adelbert Bacher
Prüfer der Dissertation: 1. apl.-Prof. Dr. Dr. h. c. Robert Huber
2. Univ.-Prof. Dr. Johannes Buchner




Die Dissertation wurde am 7.6.2004 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 28.7.2004 angenommen.
This work, although a valid scientific work, is nevertheless far from deserving an
important scientific prize, but is a very important step on my life! Unfortunately this long
step is under my personal aspirations, but is simply what was possible and nothing more. I
will take the responsibility and the consequences for this damage.
I will mainly address thanks to my supervisors, Professors Maria João Romão and Robert
Huber and also to Professor Adelbert Bacher, those who made this work possible. I also
thank some coleagues and some good friends that supported me. I am grateful to everyone
that at some stage helped a little bit to build it and even to those who not doing anything
at all, did not disturbed it.
Besides the practical knowledge that I could take from it as a “young” scientist, the most
important to me during these long years is what I learned about human society and human
nature.
I will dedicate this humble work, to two persons who always supported me
unconditionally, and those are the persons I love most in this world, my parents Maria
Helena and Manuel.
This work was financially and exclusively supported by grants PRAXIS/BM/12730/97
and PRAXISXXI/BD/21493/99 from the Fundação para a Ciência e Tecnologia /
Ministério da Ciência e Tecnologia (Portugal) and Fundo Social Europeu (III Quadro
comunitário de apoio).














2
Acknowledgments
Table of contents
Summary
Zusammenfassung
I Introduction.............................................................................................................11
1 Scope of this work..................................................................................................11
2 Protein Crystallography..........................................................................................12
2.1 Crystallization and X-ray diffraction .............................................................
2.2 Solving the phase problem .............................................................................15
2.2.1 Molecular Replacement (MR)................................................................15
2.3 Structure refinement.......................................................................................18
3 Biological pteridines...............................................................................................20
3.1 GTP cyclohydrolase I enzyme .......................................................................24
3.2 Molybdopterin containing enzyme Aldehyde oxidoreductase (AOR), as a
member of the Xanthine oxidase family. ...................................................................27
II Materials and Methods.........................................................................................29
A. GTP cyclohydrolase I from Escherischia coli ...........................................................29
II.A.Methods.1 Molecular Biology Methods ..............................................................29
II.A.Methods.1.1 Transformation in chemically competent cells..........................29
II.A.Methods.2 Protein Chemistry Methods ...............................................................29
II.A.Methods.2.1 Growth of Cells overexpressing GTP cyclohydrolase I ............29
II.A.Methods.2.2 Purification protocol ..................................................................30
II.A.Methods.2.3 Enzyme Assays..........................................................................30
II.A.Methods.2.4 Determination of zinc content....................................................30
II.A.Methods.2.5 Protein concentration measurement...........................................30
II.A.Methods.3 Crystallography Methods...................................................................31
II.A.Methods.3.1 Protein crystallization................................................................31
II.A.Methods.3.2 X-ray diffraction data collection and processing.......................31
II.A.Methods.3.3 Structure solution and refinement..............................................32
II.A.Methods.4 Software.............................................................................................32
B. The Aldehyde Oxidoreductase from Desulfovibrio gigas (MOP) .............................34
II.B.Methods.1 Protein Chemistry Methods ...............................................................34
II.B.Methods.1.1 Cell growth and protein purification..........................................34
II.B.Methods.2 Crystallography Methods...................................................................34
II.B.Methods.2.1 Protein crystallization................................................................34
II.B.Methods.2.2 X-ray diffraction and data processing........................................34
II.B.Methods.2.3 Structure Solution and Refinement ............................................35
II.B.Methods.2.4 MCD cofactor target values driven by CSD search...................39
II.B.Methods.3 Software39
C. The Aldehyde Oxidoreductase from Desulfovibrio desulfuricans 27775 (MOD).....40
II.C.Methods.1 Molecular biology methods ...............................................................40
II.C.Methods.1.1 Sequencing of the gene encoding MOD. ...................................40
II.C.Methods.2 Protein Chemistry Methods41
II.C.Methods.2.1 Protein purification ....................................................................41
II.C.Methods.3 Crystallography methods...................................................................42
II.C.Methods.3.1 Protein crystallisation.................................................................42
II.C.Methods.3.2 X-ray diffraction and data collection .........................................42
II.C.Methods.3.3 Crystallographic data processing ...............................................42
3II.C.Methods.3.4 Structure solution .......................................................................42
II.C.Methods.3.5 Model building and refinement..................................................43
II.C.Methods.4 Software.............................................................................................44
III Results and Discussion.....................................................................................46
III-A - GTP cyclohydrolase I – The complex first step of tetrahydropterin biosynthesis in
E. coli..................................................................................................................................46
III.A Results....................................................................................................................46
III.A.Results.1 Background.......................................................................................46
III.A.Results.2 Crystallographic analysis...................................................................48
III.A.Results.3 Substrate (GTP) contacts with the protein matrix..............................51
III.A.Results.4 Mutants configuration/conformation description...............................54
III.A Discussion..............................................................................................................58
III.A.Discussion.1 The reaction mechanism...............................................................58
III - B/C/D - Molybdopterin containing enzymes belonging to the Xanthine oxidase
family (Molybdenum hydroxylases). .................................................................................61
B. The Aldehyde Oxidoreductase from Desulfovibrio gigas.............................................61
III.B Results....................................................................................................................61
III.B.Results.1 Crystallisation, data collection and refinement. .................................61 .2 Overall quality of the final model ......................................................62 .3 Fourier termination effects on the Molybdenum site .........................63
III.B Discussion......................................................................................