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. 1996 Oct;5(10):2000–2008. doi: 10.1002/pro.5560051006

Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer.

R Sterner 1, G R Kleemann 1, H Szadkowski 1, A Lustig 1, M Hennig 1, K Kirschner 1
PMCID: PMC2143258  PMID: 8897600

Abstract

The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 degrees C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 degrees C, and how its thermolabile substrate, N-(5'-phosphoribosyl)-anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold beta alpha (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 degrees C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35-fold more active at its physiologic temperature than is the enzyme from E. coli (ePRAI) at 37 degrees C. This high catalytic efficiency of tPRAI is likely to complete successfully with the rapid spontaneous hydrolysis of PRA at 80 degrees C. Thus, with respect to both stability and function, tPRAI appears well adapted to the extreme habitat of T. maritima. Single crystals of tPRAI have been obtained that are suitable for X-ray analysis at high resolution.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bisswanger H., Kirschner K., Cohn W., Hager V., Hansson E. N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 1. A substrate analogue binds to two different binding sites on the bifunctional enzyme from Escherichia coli. Biochemistry. 1979 Dec 25;18(26):5946–5953. doi: 10.1021/bi00593a029. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Braus G. H., Luger K., Paravicini G., Schmidheini T., Kirschner K., Hütter R. The role of the TRP1 gene in yeast tryptophan biosynthesis. J Biol Chem. 1988 Jun 5;263(16):7868–7875. [PubMed] [Google Scholar]
  4. Bujard H., Gentz R., Lanzer M., Stueber D., Mueller M., Ibrahimi I., Haeuptle M. T., Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol. 1987;155:416–433. doi: 10.1016/0076-6879(87)55028-5. [DOI] [PubMed] [Google Scholar]
  5. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  6. Cavagnero S., Zhou Z. H., Adams M. W., Chan S. I. Response of rubredoxin from Pyrococcus furiosus to environmental changes: implications for the origin of hyperthermostability. Biochemistry. 1995 Aug 8;34(31):9865–9873. doi: 10.1021/bi00031a007. [DOI] [PubMed] [Google Scholar]
  7. Cohn W., Kirschner K., Paul C. N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analogue binds independently to two different sites. Biochemistry. 1979 Dec 25;18(26):5953–5959. doi: 10.1021/bi00593a030. [DOI] [PubMed] [Google Scholar]
  8. Crawford I. P. Evolution of a biosynthetic pathway: the tryptophan paradigm. Annu Rev Microbiol. 1989;43:567–600. doi: 10.1146/annurev.mi.43.100189.003031. [DOI] [PubMed] [Google Scholar]
  9. Creighton T. E. N-(5'-phosphoribosyl)anthranilate isomerase-indol-3-ylglycerol phosphate synthetase of tryptophan biosynthesis. Relationship between the two activities of the enzyme from Escherichia coli. Biochem J. 1970 Dec;120(4):699–707. doi: 10.1042/bj1200699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Creighton T. E. The nonenzymatic preparation in solution of N-(5'-phosphoribosyl) anthranilic acid, an intermediate in tryptophan biosynthesis. J Biol Chem. 1968 Nov 10;243(21):5605–5609. [PubMed] [Google Scholar]
  11. Creighton T. E., Yanofsky C. Indole-3-glycerol phosphate synthetase of Escherichia coli, an enzyme of the tryptophan operon. J Biol Chem. 1966 Oct 25;241(20):4616–4624. [PubMed] [Google Scholar]
  12. Darimont B., Sterner R. Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima. EMBO J. 1994 Apr 15;13(8):1772–1781. doi: 10.1002/j.1460-2075.1994.tb06445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eberhard M., Tsai-Pflugfelder M., Bolewska K., Hommel U., Kirschner K. Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains. Biochemistry. 1995 Apr 25;34(16):5419–5428. doi: 10.1021/bi00016a013. [DOI] [PubMed] [Google Scholar]
  14. Enatsu T., Crawford I. P. Enzymes of the tryptophan synthetic pathway in Pseudomonas putida. J Bacteriol. 1968 Jan;95(1):107–112. doi: 10.1128/jb.95.1.107-112.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engel A. M., Cejka Z., Lupas A., Lottspeich F., Baumeister W. Isolation and cloning of Omp alpha, a coiled-coil protein spanning the periplasmic space of the ancestral eubacterium Thermotoga maritima. EMBO J. 1992 Dec;11(12):4369–4378. doi: 10.1002/j.1460-2075.1992.tb05537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fink A. L., Calciano L. J., Goto Y., Kurotsu T., Palleros D. R. Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry. 1994 Oct 18;33(41):12504–12511. doi: 10.1021/bi00207a018. [DOI] [PubMed] [Google Scholar]
  17. Fontana A. Structure and stability of thermophilic enzymes. Studies on thermolysin. Biophys Chem. 1988 Feb;29(1-2):181–193. doi: 10.1016/0301-4622(88)87038-8. [DOI] [PubMed] [Google Scholar]
  18. Hass M, Ahmad I, I, Janssens RV, Khoo TL, Körner HJ, Moore EF, Wolfs FH, Benczer-Koller N, Dafni E, Beard K. g factor of the (59/2(-) isomer in 147Gd. Phys Rev C Nucl Phys. 1989 Jun;39(6):2237–2241. doi: 10.1103/physrevc.39.2237. [DOI] [PubMed] [Google Scholar]
  19. Hess D., Krüger K., Knappik A., Palm P., Hensel R. Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus. Eur J Biochem. 1995 Oct 1;233(1):227–237. doi: 10.1111/j.1432-1033.1995.227_1.x. [DOI] [PubMed] [Google Scholar]
  20. Hoch S. O., Anagnostopoulos C., Crawford I. P. Enzymes of the tryptophan operon of Bacillus subtilis. Biochem Biophys Res Commun. 1969 Jun 27;35(6):838–844. doi: 10.1016/0006-291x(69)90700-1. [DOI] [PubMed] [Google Scholar]
  21. Hommel U., Eberhard M., Kirschner K. Phosphoribosyl anthranilate isomerase catalyzes a reversible amadori reaction. Biochemistry. 1995 Apr 25;34(16):5429–5439. doi: 10.1021/bi00016a014. [DOI] [PubMed] [Google Scholar]
  22. Hommel U., Lustig A., Kirschner K. Purification and characterization of yeast anthranilate phosphoribosyltransferase. Eur J Biochem. 1989 Mar 1;180(1):33–40. doi: 10.1111/j.1432-1033.1989.tb14611.x. [DOI] [PubMed] [Google Scholar]
  23. Jackson E. N., Yanofsky C. Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function. J Mol Biol. 1973 May 5;76(1):89–101. doi: 10.1016/0022-2836(73)90082-x. [DOI] [PubMed] [Google Scholar]
  24. Jaenicke R. Stability and folding of ultrastable proteins: eye lens crystallins and enzymes from thermophiles. FASEB J. 1996 Jan;10(1):84–92. doi: 10.1096/fasebj.10.1.8566552. [DOI] [PubMed] [Google Scholar]
  25. Kirschner K., Szadkowski H., Jardetzky T. S., Hager V. Phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthase from Escherichia coli. Methods Enzymol. 1987;142:386–397. doi: 10.1016/s0076-6879(87)42050-8. [DOI] [PubMed] [Google Scholar]
  26. Kohlhoff M., Dahm A., Hensel R. Tetrameric triosephosphate isomerase from hyperthermophilic Archaea. FEBS Lett. 1996 Apr 1;383(3):245–250. doi: 10.1016/0014-5793(96)00249-9. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Larralde R., Robertson M. P., Miller S. L. Rates of decomposition of ribose and other sugars: implications for chemical evolution. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8158–8160. doi: 10.1073/pnas.92.18.8158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levine R. L., Federici M. M. Quantitation of aromatic residues in proteins: model compounds for second-derivative spectroscopy. Biochemistry. 1982 May 25;21(11):2600–2606. doi: 10.1021/bi00540a004. [DOI] [PubMed] [Google Scholar]
  30. Martins L. O., Santos H. Accumulation of Mannosylglycerate and Di-myo-Inositol-Phosphate by Pyrococcus furiosus in Response to Salinity and Temperature. Appl Environ Microbiol. 1995 Sep;61(9):3299–3303. doi: 10.1128/aem.61.9.3299-3303.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ostendorp R., Liebl W., Schurig H., Jaenicke R. The L-lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli. Eur J Biochem. 1993 Sep 15;216(3):709–715. doi: 10.1111/j.1432-1033.1993.tb18190.x. [DOI] [PubMed] [Google Scholar]
  32. Priestle J. P., Grütter M. G., White J. L., Vincent M. G., Kania M., Wilson E., Jardetzky T. S., Kirschner K., Jansonius J. N. Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5690–5694. doi: 10.1073/pnas.84.16.5690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reardon D., Farber G. K. The structure and evolution of alpha/beta barrel proteins. FASEB J. 1995 Apr;9(7):497–503. doi: 10.1096/fasebj.9.7.7737457. [DOI] [PubMed] [Google Scholar]
  34. Sanangelantoni A. M., Forlani G., Ambroselli F., Cammarano P., Tiboni O. The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli. J Gen Microbiol. 1992 Feb;138(2):383–393. doi: 10.1099/00221287-138-2-383. [DOI] [PubMed] [Google Scholar]
  35. Scholz S., Sonnenbichler J., Schäfer W., Hensel R. Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei. FEBS Lett. 1992 Jul 20;306(2-3):239–242. doi: 10.1016/0014-5793(92)81008-a. [DOI] [PubMed] [Google Scholar]
  36. Schurig H., Beaucamp N., Ostendorp R., Jaenicke R., Adler E., Knowles J. R. Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex. EMBO J. 1995 Feb 1;14(3):442–451. doi: 10.1002/j.1460-2075.1995.tb07020.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Srivastava D. K., Smolen P., Betts G. F., Fukushima T., Spivey H. O., Bernhard S. A. Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation? Proc Natl Acad Sci U S A. 1989 Sep;86(17):6464–6468. doi: 10.1073/pnas.86.17.6464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sterner R., Dahm A., Darimont B., Ivens A., Liebl W., Kirschner K. (Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima. EMBO J. 1995 Sep 15;14(18):4395–4402. doi: 10.1002/j.1460-2075.1995.tb00118.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stoll V. S., Blanchard J. S. Buffers: principles and practice. Methods Enzymol. 1990;182:24–38. doi: 10.1016/0076-6879(90)82006-n. [DOI] [PubMed] [Google Scholar]
  40. Tomschy A., Glockshuber R., Jaenicke R. Functional expression of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli. Authenticity and kinetic properties of the recombinant enzyme. Eur J Biochem. 1993 May 15;214(1):43–50. doi: 10.1111/j.1432-1033.1993.tb17894.x. [DOI] [PubMed] [Google Scholar]
  41. Vieille C., Hess J. M., Kelly R. M., Zeikus J. G. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol. 1995 May;61(5):1867–1875. doi: 10.1128/aem.61.5.1867-1875.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wilmanns M., Priestle J. P., Niermann T., Jansonius J. N. Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution. J Mol Biol. 1992 Jan 20;223(2):477–507. doi: 10.1016/0022-2836(92)90665-7. [DOI] [PubMed] [Google Scholar]
  43. Winterhalter C., Heinrich P., Candussio A., Wich G., Liebl W. Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol. 1995 Feb;15(3):431–444. doi: 10.1111/j.1365-2958.1995.tb02257.x. [DOI] [PubMed] [Google Scholar]
  44. Woese C. R., Kandler O., Wheelis M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. doi: 10.1073/pnas.87.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wrba A., Jaenicke R., Huber R., Stetter K. O. Lactate dehydrogenase from the extreme thermophile Thermotoga maritima. Eur J Biochem. 1990 Feb 22;188(1):195–201. doi: 10.1111/j.1432-1033.1990.tb15388.x. [DOI] [PubMed] [Google Scholar]
  46. Yaylayan V. A., Huyghues-Despointes A. Chemistry of Amadori rearrangement products: analysis, synthesis, kinetics, reactions, and spectroscopic properties. Crit Rev Food Sci Nutr. 1994;34(4):321–369. doi: 10.1080/10408399409527667. [DOI] [PubMed] [Google Scholar]

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