Conclusions

and especially the oxyanion. On the other hand, it is possible that the formation of the formyl-aspartic anhydride occurs by concerted mechanism, which would lack the tetrahedral intermediate and in turn no oxyanion would be formed. This question can be addressed by ¹³C and ¹⁸O isotope effect measurements.

Finally the question of whether Frc and Ocdc interact forming a stable complex has to be addressed. As mentioned above, formyl-CoA is a very unstable thioester easily susceptible to hydrolysis (paper III), producing CoA and formate which are both Frc inhibitors (Jonsson, unpublished results). So hydrolysis of formyl-CoA is not only degrading the substrate of Frc but also it releases Frc inhibitors. Therefore it is feasible to think that Frc and Ocdc would form a complex in order to protect formyl-CoA from hydrolysis. Moreover two members of family III have been reported to take part in large enzyme complexes: (R)-phenyllactate-CoA transferase is part of phenyllactate dehydratase, a heterotrimeric complex (Dickert et al., 2000) and L-carnitine CoA transferase is a component of the two protein complex, crotonobetaine reductase (Preusser et al., 1999).

3 REFERENCES

Allison, M.J. and Cook, H.M. (1981) Oxalate degradation by microbes of the large bowel of herbivores: the effect of dietary oxalate. Science, 212, 675-676.

Allison, M.J., Dawson, K.A., Mayberry, W.R. and Foss, J.G. (1985) Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading

anaerobes that inhabit the gastrointestinal tract. Arch Microbiol, 141, 1-7.

Baetz, A.L. and Allison, M.J. (1989) Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes. J Bacteriol, 171, 2605-2608.

Baetz, A.L. and Allison, M.J. (1990) Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes. J Bacteriol, 172, 3537-3540.

Barker, H.A., Jeng, I.M., Neff, N., Robertson, J.M., Tam, F.K. and Hosaka, S.

(1978) Butyryl-CoA:acetoacetate CoA-transferase from a lysine-fermenting Clostridium. J Biol Chem, 253, 1219-1225.

Bateman, K.S., Brownie, E.R., Wolodko, W.T. and Fraser, M.E. (2002) Structure of the mammalian CoA transferase from pig heart.

Biochemistry, 41, 14455-14462.

Boggon, T.J. and Shapiro, L. (2000) Screening for phasing atoms in protein crystallography. Structure Fold Des, 8, R143-149.

Buckel, W. and Bobi, A. (1976) The enzyme complex citramalate lyase from Clostridium tetanomorphum. Eur J Biochem, 64, 255-262.

Buckel, W., Dorn, U. and Semmler, R. (1981) Glutaconate CoA-transferase from Acidaminococcus fermentans. Eur J Biochem, 118, 315-321.

Buckley, P.A., Baz Jackson, J., Schneider, T., White, S.A., Rice, D.W. and Baker, P.J. (2000) Protein-protein recognition, hydride transfer and proton pumping in the transhydrogenase complex. Structure Fold Des, 8, 809-815.

Carey, J. (2000) A systematic and general proteolytic method for defining structural and functional domains of proteins. Methods Enzymol, 328, 499-514.

Clark, J.H., Fitzgerald, J.F. and Bergstein, J.M. (1985) Nephrolithiasis in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr, 4, 829-834.

Clements, A., Rojas, J.R., Trievel, R.C., Wang, L., Berger, S.L. and Marmorstein, R. (1999) Crystal structure of the histone

acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A. Embo J, 18, 3521-3532.

Cornick, N.A. and Allison, M.J. (1996) Assimilation of oxalate, acetate, and CO2 by Oxalobacter formigenes. Can J Microbiol, 42, 1081-1086.

Corthesy-Theulaz, I.E., Bergonzelli, G.E., Henry, H., Bachmann, D., Schorderet, D.F., Blum, A.L. and Ornston, L.N. (1997) Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

J Biol Chem, 272, 25659-25667.

Dawson, K.A., Allison, M.J. and Hartman, P.A. (1980) Isolation and some characteristics of anaerobic oxalate-degrading bacteria from the

Dickert, S., Pierik, A.J., Linder, D. and Buckel, W. (2000) The involvement of coenzyme A esters in the dehydration of (R)-phenyllactate to (E)-cinnamate by Clostridium sporogenes. Eur J Biochem, 267, 3874-3884.

Dimroth, P. and Eggerer, H. (1975) Isolation of subunits of citrate lyase and characterization of their function in the enzyme complex. Proc Natl Acad Sci U S A, 72, 3458-3462.

Doublie, S. (1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol, 276, 523-530.

Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B. and Jordan, F.

(1993) Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution. Biochemistry, 32, 6165-6170.

Elssner, T., Engemann, C., Baumgart, K. and Kleber, H.P. (2001) Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli. Biochemistry, 40, 11140-11148.

Engel, C. and Wierenga, R. (1996) The diverse world of coenzyme A binding proteins. Curr Opin Struct Biol, 6, 790-797.

Gogos, A., Gorman, J. and Shapiro, L. (2004) Structure of Escherichia coli YfdW, a type III CoA transferase. Acta Crystallogr D Biol Crystallogr, 60, 507-511.

Gruez, A., Roig-Zamboni, V., Valencia, C., Campanacci, V. and Cambillau, C.

(2003) The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases. J Biol Chem, 278, 34582-34586.

Heider, J. (2001) A new family of CoA-transferases. FEBS Lett, 509, 345-349.

Heymann, J.A., Hirai, T., Shi, D. and Subramaniam, S. (2003) Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution. J Struct Biol, 144, 320-326.

Hirai, T., Heymann, J.A., Shi, D., Sarker, R., Maloney, P.C. and

Subramaniam, S. (2002) Three-dimensional structure of a bacterial oxalate transporter. Nat Struct Biol, 9, 597-600.

Hodgkinson, A. (1977) Oxalic acid in Biology and in Medicine. Academic press, London.

Hylander, E., Jarnum, S., Jensen, H.J. and Thale, M. (1978) Enteric hyperoxaluria: dependence on small intestinal resection, colectomy, and steatorrhoea in chronic inflammatory bowel disease. Scand J Gastroenterol, 13, 577-588.

Jacob, U., Mack, M., Clausen, T., Huber, R., Buckel, W. and Messerschmidt, A. (1997) Glutaconate CoA-transferase from Acidaminococcus

fermentans: the crystal structure reveals homology with other CoA-transferases. Structure, 5, 415-426.

Johansson, E., Steffens, J.J., Lindqvist, Y. and Schneider, G. (2000) Crystal structure of saccharopine reductase from Magnaporthe grisea, an enzyme of the alpha-aminoadipate pathway of lysine biosynthesis.

Structure Fold Des, 8, 1037-1047.

Joyce, M.A., Fraser, M.E., James, M.N., Bridger, W.A. and Wolodko, W.T.

(2000) ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by x-ray crystallography. Biochemistry, 39, 17-25.

Leuthner, B. and Heider, J. (2000) Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate. J Bacteriol, 182, 272-277.

Leutwein, C. and Heider, J. (2001) Succinyl-CoA:(R)-benzylsuccinate CoA-transferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria. J Bacteriol, 183, 4288-4295.

Lung, H.Y., Baetz, A.L. and Peck, A.B. (1994) Molecular cloning, DNA

sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes. J Bacteriol, 176, 2468-2472.

Mahler, H.R. and Cordes, E.H. (1966) Biological Chemistry. Harper, Row &

Weatherhill, New York.

McGilvery, R.W. (1970) Biochemistry: A Pratical Approach. WB Saunders Company, Philadelphia.

Nagem, R.A., Dauter, Z. and Polikarpov, I. (2001) Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers. Acta Crystallogr D Biol Crystallogr, 57, 996-1002.

Parales, R.E. and Harwood, C.S. (1992) Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida. J Bacteriol, 174, 4657-4666.

Pintacuda, G., Moshref, A., Leonchicks, A., Sharipo, A. and Otting, G. (2004) Site-specific labelling with a metal chelator for protein-structure refinement. Journal of Biomolecular NMR, In press.

Preusser, A., Wagner, U., Elssner, T. and Kleber, H.P. (1999) Crotonobetaine reductase from Escherichia coli consists of two proteins. Biochim Biophys Acta, 1431, 166-178.

Purdy, M.D., Ge, P., Chen, J., Selvin, P.R. and Wiener, M.C. (2002) Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.

Acta Crystallogr D Biol Crystallogr, 58, 1111-1117.

Rodby, R.A., Tyszka, T.S. and Williams, J.W. (1991) Reversal of cardiac

dysfunction secondary to type 1 primary hyperoxaluria after combined liver-kidney transplantation. Am J Med, 90, 498-504.

Ruan, Z.S., Anantharam, V., Crawford, I.T., Ambudkar, S.V., Rhee, S.Y., Allison, M.J. and Maloney, P.C. (1992) Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of

Oxalobacter formigenes. J Biol Chem, 267, 10537-10543.

Saraste, M., Sibbald, P.R. and Wittinghofer, A. (1990) The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci, 15, 430-434.

Schulz, G. (1992) Binding of nucleotides by proteins. Curr Opin Struct Biol, 2, 61-67.

Selmer, T. and Buckel, W. (1999) Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans. Implications for the mechanism of CoA-ester hydrolysis. J Biol Chem, 274, 20772-20778.

Sidhu, H., Hoppe, B., Hesse, A., Tenbrock, K., Brömme, S., Rietschel, E. and Peck, A.B. (1998) Absence of oxalobacter formigenes in cystic fibrosis patients: a risk factor for hyperoxaluria. Lancet, 352, 1026-1029.

Sidhu, H., Ogden, S.D., Lung, H.Y., Luttge, B.G., Baetz, A.L. and Peck, A.B.

(1997) DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes. J Bacteriol, 179, 3378-3381.

Sidhu, H., Schmidt, M.E., Cornelius, J.G., Thamilselvan, S., Khan, S.R., Hesse, A. and Peck, A.B. (1999) Direct correlation between

hyperoxaluria/oxalate stone disease and the absence of the

gastrointestinal tract-dwelling bacterium Oxalobacter formigenes:

possible prevention by gut recolonization or enzyme replacement therapy. J Am Soc Nephrol, 10 Suppl 14, S334-340.

Stewart, C.S., Duncan, S.H. and Cave, D.R. (2004) Oxalobacter formigenes and its role in oxalate metabolism in the human gut. FEMS Microbiol Lett, 230, 1-7.

Terwilliger, T.C. and Berendzen, J. (1999) Automated MAD and MIR structure solution. Acta Crystallogr D Biol Crystallogr, apr 55, 849-861.

Troxel, S.A., Sidhu, H., Kaul, P. and Low, R.K. (2003) Intestinal Oxalobacter formigenes colonization in calcium oxalate stone formers and its relation to urinary oxalate. J Endourol, 17, 173-176.

White, H. and Jencks, W.P. (1976) Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase. J Biol Chem, 251, 1688-1699.

Wierenga, R.K., Terpstra, P. and Hol, W.G. (1986) Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol, 187, 101-107.

Wiesenborn, D.P., Rudolph, F.B. and Papoutsakis, E.T. (1989) Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids. Appl Environ Microbiol, 55, 323-329.

Williams, H.E. and Smith, L.H., Jr. (1968) Disorders of oxalate metabolism.

Am J Med, 45, 715-735.

Wolf, E., Vassilev, A., Makino, Y., Sali, A., Nakatani, Y. and Burley, S.K.

(1998) Crystal structure of a GCN5-related N-acetyltransferase:

Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell, 94, 439-449.

Wolodko, W.T., Fraser, M.E., James, M.N. and Bridger, W.A. (1994) The crystal structure of succinyl-CoA synthetase from Escherichia coli at 2.5-A resolution. J Biol Chem, 269, 10883-10890.

4 ACKNOWLEDGEMENTS

I would like to thank everyone who contributed in any way to the work here presented.

In particular I need to thank:

My main supervisor, Ylva Lindqvist, for giving me the possibility to carry out my PhD studies in this laboratory; for being very knowledgeable, enthusiastic about the project, patient and supportive.

My second supervisor Gunter Schneider, he is a very good group leader and a skilful cook. I enjoyed a lot both his abilities. I want to thank him particularly for his guidance and support in some difficult situations.

Prof. Nigel Richards and Stefan Jonsson from Dept. of Chemistry, University of Florida for the very fruitful collaboration over the years. Thank to

Stefan for showing why from the weather point of view Florida is a better choice than Stockholm.

Prof. Jannette Carey and Dr. Williams Peters from Dept. of Chemistry at Princeton University for very interesting collaboration although I had unfortunately very limited time to explore the potential of our common project.

Anja Schütz, the sweetest collaborator a person can hope to have!

Now it is time to thank all present and former members of Molecular Structural Biology lab. Few months after I arrived in Sweden Eva came to me saying "I saw a program on TV about the italian "mammoni": you are italian and you need a mother taking care of you, I will be your swedish mother." So she said and so she did, I enormously thankful for that.

Doreen, I don't know exactly for how many things I need to thank you, for being a good friend over the years, a good crystallography teacher, a good listener of my moaning, an example not to follow in learning swedish... Is it enough?

I guess I go on in mentioning the recently formed boy band, good

companions in and outside the lab: Bernie who spent some time with my difficult refinements, office mate, who taught me what people mean in Scotland by being hard drinkers. The cool new PhD student, Daniel, the perfect mix of viking macho and city boy, I expect a lot from you. Bad boy Philipp, always critic but always great fun, he even complains when I kick him playingfootball. I need to thank Robert especially for his pervert jokes.

Another key member of the boy band does not belong to the lab but this the proper place to mention Masatoshi Nukui San.

I shared all my PhD time with Anna, we went to conferences, climbing, motorbike together it was very nice time.

I had few students in these years, my favourite was definitely Catrine; now that she started as a PhD student in the lab and I am happy to leave the oxalate project in her good hands.

Tanja our synchrotron master, the person who can work day and night at the beam line without sleep, with an apple for lunch and dinner, without becoming grumpy and irritated, thank you for your help, support and example.

...and a lot of other people the sweet Azmiri, Stina L., Mona, Jodie checking with great patience my italiano/english thesis and Magnus nice people always available to help or simply for a chat.

Let's mention then some heroes from the glorious old days in the lab.

Martin, you showed me how Swedes enjoy their days: being in Globen for a hockey match, outside at -20 C ° watching 22 bandy players playing with an invisible tiny ball, drinking quickly a beer in a sauna before the beer starts to boil.... Jenny, a good lunch mate, nice conversations over three years time: everything around you has to be good and perfect, I am thankful that you accepted me as a friend anyway. Lucas my office mate for more than 3 years. Matthias a former post-doc in the lab, he is quite a character. Susana, we shared the mediterranean attitude in the lab.

Adnane, it is not easy to be friend with him, he takes things too personally but I rarely experienced a more volcanic humour. Stina T. told me once: "a PhD student has to suffer!" I think I got my share, but let me disagree: it was also a lot of fun. And more people Louise and Eva J. Alexei, Jessy and Alokesh who showed me how weird a person can be.

In MBB our little italian network: Guido genial, confused, nice friend as knowledgeable about NMR as about football and Simone, in how many party have we been together?

A lot of other people in the department I spent great time with: Anna K. a brave motorbike companion, Cecilia, Anna P., Anna, Jessica, Hans, Andis, Jakko, Marko, Joha and lot of others ....

Out of MBB it is even more difficult to remember all the people that made great these years in Stockholm.

Ainky the first who introduced me to the Eriksson social life, one of the best P.R. that Stockholm has experienced, deep person, good friend.

The italian group: Prof. Lol— Richiardi wonderful friend, his place will be my "pied-‡-terre" in Italy as soon as move to Marseille, I thank you already