Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 332 Structural and Functional Studies of Ribose-5-phosphate isomerase B ANNETTE K. ROOS ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-6952-8 2007 urn:nbn:se:uu:diva-8182 ! ""# $%"" & ' & & (' ') *' + , ') - . /) ""#) & - 010 ' ' ) . ) ) # ) ) 2 3 4#!04$01105410!) - 10 ' ' 6- 7 & ' 8 9 & ' ' ' '+ +' ' 0 & 10 ' ' 6-1(7 10 ' ') *+ & & ' +' & : - . - ) *' ' - & ' 67 67 & & & +) ' ' - . - +'' ' ' - ' && 050 ' ' ) . ' ' + ' - ' ' ) 2 ' ' & - ) *' & - + +' && ' ' + +' ' -1() *' & ' ' ' ' & - & ' - ) . & - ' + ' ' -1( ' 50 ' ' ) && ' + - - ' ; ) . & - +' ' 44 + ' ' - ' & & ' +'' ' & ' + ' ) 2 ' ' ' ' - - . & ' +'' ' ' ' - .) && ' ' & ' + < - & & & ' +' ' & + 0 ) ! ' 0 '' 050 ' ' ' ' '+ -51 =0 ' " # $ % $ % & '()$ $ *+',-. $ > . /) - ""# 2 3 $51$05$ 2 3 4#!04$01105410! % %%% 0!$! 6' %?? );)? @ A % %%% 0!$!7 List of Papers This thesis is a summary of the results presented in the following publica- tions and manuscript, which will be referred to by their roman numerals: I Roos A. K., Andersson C. E., Bergfors T., Jacobsson M., Karlen A., Unge T., Jones T. A., Mowbray S. L. (2004) My- cobacterium tuberculosis Ribose-5-phosphate Isomerase has a known fold, but a novel active site. J. Mol. Biol. 335, 799- 809. II Roos A. K., Burgos E., Ericsson D. J., Salmon L., Mowbray S. L. (2005) Competitive Inhibitors of Mycobacterium tuber- culosis Ribose-5-phosphate Isomerase B Reveal New Infor- mation About the Reaction Mechanism. J. Biol. Chem. 280, 6416-6422. III Burgos E., Roos A. K., Mowbray S. L., Salmon L. (2005) Synthesis of 5-deoxy-5-phospho-D-ribonohydroxamic acid: a new competitive and selective inhibitor of type B ribose-5- phosphate isomerase from Mycobacterium tuberculosis. Tet- rahedron Lett. 46, 3691-3694. IV Roos A. K., Mariano S., Kowalinski E., Salmon L., Mow- bray S. L. (2007) Ribose-5-phosphate isomerase B from Es- cherichia coli is also a functional allose-6-phosphate isom- erase while the Mycobacterium tuberculosis enzyme is not. Manuscript Articles I-III are printed with permission from the copyright holders. iii Additional Paper Nurbo J., Roos A. K., Muthas D., Wahlström E., Ericsson D. J., Lunstedt T., Unge T., Karlen A. (2007) Design, Synthesis and Evaluation of Peptide In- hibitors of Mycobacterium tuberculosis Ribonucleotide Reductase. J. Peptide Science, In press iv Contents Introduction.....................................................................................................1 Conveying ribose across the cell membrane ..............................................1 The Pentose Phosphate Pathway ................................................................2 Ribose-5-phosphate isomerase – introducing the main character ..............4 The als operon of E. coli ............................................................................7 Mycobacterium tuberculosis ....................................................................10 Sugar transport in M. tuberculosis.......................................................10 The pentose phosphate pathway of M. tuberculosis............................11 General considerations in designing new antitubercular drugs................12 Rpi essentiality studies in bacteria and fungi.......................................12 A human Rpi mutant causing abnormal brain development................13 Rpi – a possible drug target? ...............................................................14 Current investigation.....................................................................................15 Aim of thesis ............................................................................................15 Introductory sequence comparisons and genomics searches....................16 Functional studies of MtRpiB and EcRpiB ..............................................18 Assay results of MtRpiB......................................................................20 Assay results of EcRpiB ......................................................................25 Allose-6-phosphate isomerase activity (Paper IV) ..............................26 Crystallisation...........................................................................................27 Structural results.......................................................................................33 Solving the structure of MtRpiB (Paper I)...........................................33 The overall structure of MtRpiB..........................................................35 The active site of MtRpiB....................................................................37 E. coli RpiB structures.........................................................................42 Comparing the Ec- and MtRpiB structures ..............................................48 The proposed reaction mechanism of RpiB .............................................51 The reaction as catalysed by RpiA...........................................................54 Conclusions and future perspectives.............................................................56 Summary in Swedish ....................................................................................59 Acknowledgements.......................................................................................64 References.....................................................................................................68 v Abbreviations Rpi Ribose-5-phosphate isomerase Mt / M. tuberculosis Mycobacterium tuberculosis Ec / E. coli Escherichia coli PPP Pentose phosphate pathway R5P Ribose 5-phosphate Ru5P Ribulose 5-phosphate All6P Allose 6-phosphate Allu6P Allulose 6-phosphate 4PEA 4-deoxy-4-phospho-D-erythronate 4PEH 4-deoxy-4-phospho-D-erythronohydroxamic acid 4PEAm 4-deoxy-4-phospho-D-erythronamide 4PEHz 4-deoxy-4-phospho-D-erythronhydrazide 4PMEA 4-deoxy-4-phosphonomethyl-D-erythronate 5PRH 5-deoxy-5-phospho-D-ribonohydroxamic acid 5PRA 5-deoxy-5-phospho-D-ribonate Pi Inorganic phosphate F6P Fructose 6-phosphate G6P Glucose 6-phosphate PGI Phosphoglucose isomerase TIM Triose-phosphate isomerase MESNA 2-mercaptoethanesulfonic acid ME -mercaptoethanol MES 4-morpholineethanesulfonic acid HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonate PDB Protein Data Bank MR molecular replacement CC-F correlation coefficient AU asymmetric unit NCS non-crystallographic symmetry r.m.s. root-mean-square vi Introduction All organisms need energy to be able to exist. Some, like plants, get their energy from the sun through the process called photosynthesis. Others, like animals, birds and most bacteria, have to oxidise organic compounds in or- der to survive. These organic compounds are processed by different enzymes in metabolic pathways. In such pathways, the organic compounds are either rearranged and put together to become larger molecules, or rearranged and disassembled to become smaller building blocks, often releasing energy in the process. This thesis deals with an enzyme involved in one of the central metabolic pathways, the pentose phosphate pathway. The enzyme is called ribose-5-phosphate isomerase and the main metabolite it creates is ribose 5- phosphate. Ribose-5-phosphate isomerase has been studied in two bacterial species, Escherichia coli and Mycobacterium tuberculosis. E. coli was one of the first model organisms employed by biological researchers and many of its main metabolic pathways have now been thoroughly characterised. One such pathway is that needed for utilising ribose. Conveying ribose across the cell membrane Bacterial cells often use so-called ABC transporters as a means to actively carry small molecules through the hydrophobic cell membrane into the cyto- plasm. The three components of these transporters include a periplasmic binding protein, which has selective affinity to the recognised ligand, a membrane bound protein that is the actual carrier, and an ATP binding pro- tein, which provides the uptake system with energy. For the sugar ribose, E. coli has a high affinity transport system encoded by the genes in the rbsDACBK operon, which has been located to 84 min on the E. coli genetic map (Iida et al., 1984; Lopilato et al., 1984). The actual transport is taken care of by the rbsACB gene products: RbsA a cytoplasmic ATPase, RbsC a membrane permease and RbsB the periplasmic ribose bind- ing protein (Figure 1). RbsD is a mutarotase, that converts the pyranose form of ribose to the furanose form (Ryu et al., 2004). This is thought to help the last gene product of the operon, RbsK or ribokinase, whose job is to phosphorylate ribose. The structure of ribokinase shows that the enzyme binds the -furanose form of the sugar, a form less common in solution 1 compared to the - and -pyranoses (Sigrell et al., 1998). Once ribose is phosphorylated it is trapped within the cell due to the addition of a negative charge, which prevents it from leaking back out across the membrane. Ri- bose 5-phosphate (R5P) now enters the pentose phosphate pathway or is used for building cellular metabolites. The Pentose Phosphate Pathway The pentose phosphate pathway (PPP), also