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. 1995 May;4(5):994–1000. doi: 10.1002/pro.5560040519

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

M L Vignais 1, C Corbier 1, G Mulliert 1, C Branlant 1, G Branlant 1
PMCID: PMC2143130  PMID: 7663355

Abstract

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.

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Selected References

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  1. Biesecker G., Harris J. I., Thierry J. C., Walker J. E., Wonacott A. J. Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus. Nature. 1977 Mar 24;266(5600):328–333. doi: 10.1038/266328a0. [DOI] [PubMed] [Google Scholar]
  2. Buchwalder A., Szadkowski H., Kirschner K. A fully active variant of dihydrofolate reductase with a circularly permuted sequence. Biochemistry. 1992 Feb 18;31(6):1621–1630. doi: 10.1021/bi00121a006. [DOI] [PubMed] [Google Scholar]
  3. Charpentier B., Branlant C. The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32. J Bacteriol. 1994 Feb;176(3):830–839. doi: 10.1128/jb.176.3.830-839.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clermont S., Corbier C., Mely Y., Gerard D., Wonacott A., Branlant G. Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold. Biochemistry. 1993 Sep 28;32(38):10178–10184. doi: 10.1021/bi00089a038. [DOI] [PubMed] [Google Scholar]
  5. Corbier C., Clermont S., Billard P., Skarzynski T., Branlant C., Wonacott A., Branlant G. Probing the coenzyme specificity of glyceraldehyde-3-phosphate dehydrogenases by site-directed mutagenesis. Biochemistry. 1990 Jul 31;29(30):7101–7106. doi: 10.1021/bi00482a022. [DOI] [PubMed] [Google Scholar]
  6. Corbier C., Michels S., Wonacott A. J., Branlant G. Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification. Biochemistry. 1994 Mar 22;33(11):3260–3265. doi: 10.1021/bi00177a017. [DOI] [PubMed] [Google Scholar]
  7. Ferdinand W. The isolation and specific activity of rabbit-muscle glyceraldehyde phosphate dehydrogenase. Biochem J. 1964 Sep;92(3):578–585. doi: 10.1042/bj0920578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldenberg D. P., Creighton T. E. Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor. J Mol Biol. 1983 Apr 5;165(2):407–413. doi: 10.1016/s0022-2836(83)80265-4. [DOI] [PubMed] [Google Scholar]
  9. Hahn M., Piotukh K., Borriss R., Heinemann U. Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10417–10421. doi: 10.1073/pnas.91.22.10417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kreitman R. J., Puri R. K., Pastan I. A circularly permuted recombinant interleukin 4 toxin with increased activity. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6889–6893. doi: 10.1073/pnas.91.15.6889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Luger K., Hommel U., Herold M., Hofsteenge J., Kirschner K. Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo. Science. 1989 Jan 13;243(4888):206–210. doi: 10.1126/science.2643160. [DOI] [PubMed] [Google Scholar]
  13. Moras D., Olsen K. W., Sabesan M. N., Buehner M., Ford G. C., Rossmann M. G. Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1975 Dec 10;250(23):9137–9162. doi: 10.2210/pdb1gpd/pdb. [DOI] [PubMed] [Google Scholar]
  14. Mougin A., Corbier C., Soukri A., Wonacott A., Branlant C., Branlant G. Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase. Protein Eng. 1988 Apr;2(1):45–48. doi: 10.1093/protein/2.1.45. [DOI] [PubMed] [Google Scholar]
  15. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  16. Rossmann M. G., Adams M. J., Buehner M., Ford G. C., Hackert M. L., Liljas A., Rao S. T., Banaszak L. J., Hill E., Tsernoglou D. Letter: Molecular symmetry axes and subunit interfaces in certain dehydrogenases. J Mol Biol. 1973 Jun 5;76(4):533–537. doi: 10.1016/0022-2836(73)90491-9. [DOI] [PubMed] [Google Scholar]
  17. Skarzyński T., Moody P. C., Wonacott A. J. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution. J Mol Biol. 1987 Jan 5;193(1):171–187. doi: 10.1016/0022-2836(87)90635-8. [DOI] [PubMed] [Google Scholar]
  18. Suzuki K., Imahori K. Glyceraldehyde 3-phosphate dehydrogenase of Bacillus stearothermophilus. Kinetics and physicochemical studies. J Biochem. 1973 Nov;74(5):955–970. [PubMed] [Google Scholar]
  19. Yang Y. R., Schachman H. K. Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11980–11984. doi: 10.1073/pnas.90.24.11980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yarbrough P. O., Hayden M. A., Dunn L. A., Vermersch P. S., Klass M. R., Hecht R. M. The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes. Biochim Biophys Acta. 1987 Jan 28;908(1):21–33. doi: 10.1016/0167-4781(87)90018-2. [DOI] [PubMed] [Google Scholar]
  21. Zhang T., Bertelsen E., Benvegnu D., Alber T. Circular permutation of T4 lysozyme. Biochemistry. 1993 Nov 23;32(46):12311–12318. doi: 10.1021/bi00097a006. [DOI] [PubMed] [Google Scholar]

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