Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Apr 15;291(Pt 2):575–583. doi: 10.1042/bj2910575

Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.

L Varsani 1, T Cui 1, M Rangarajan 1, B S Hartley 1, J Goldberg 1, C Collyer 1, D M Blow 1
PMCID: PMC1132563  PMID: 8484737

Abstract

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

Full text

PDF
575

Images in this article

575Figure 1
on p.575

Selected References

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

  1. Amore R., Hollenberg C. P. Xylose isomerase from Actinoplanes missouriensis: primary structure of the gene and the protein. Nucleic Acids Res. 1989 Sep 25;17(18):7515–7515. doi: 10.1093/nar/17.18.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carrell H. L., Glusker J. P., Burger V., Manfre F., Tritsch D., Biellmann J. F. X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4440–4444. doi: 10.1073/pnas.86.12.4440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carrell H. L., Rubin B. H., Hurley T. J., Glusker J. P. X-ray crystal structure of D-xylose isomerase at 4-A resolution. J Biol Chem. 1984 Mar 10;259(5):3230–3236. [PubMed] [Google Scholar]
  4. Collyer C. A., Henrick K., Blow D. M. Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift. J Mol Biol. 1990 Mar 5;212(1):211–235. doi: 10.1016/0022-2836(90)90316-E. [DOI] [PubMed] [Google Scholar]
  5. Creighton T. E. Electrophoretic analysis of the unfolding of proteins by urea. J Mol Biol. 1979 Apr 5;129(2):235–264. doi: 10.1016/0022-2836(79)90279-1. [DOI] [PubMed] [Google Scholar]
  6. Farber G. K., Petsko G. A., Ringe D. The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes. Protein Eng. 1987 Dec;1(6):459–466. doi: 10.1093/protein/1.6.459. [DOI] [PubMed] [Google Scholar]
  7. Henrick K., Collyer C. A., Blow D. M. Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively. J Mol Biol. 1989 Jul 5;208(1):129–157. doi: 10.1016/0022-2836(89)90092-2. [DOI] [PubMed] [Google Scholar]
  8. Jenkins J., Janin J., Rey F., Chiadmi M., van Tilbeurgh H., Lasters I., De Maeyer M., Van Belle D., Wodak S. J., Lauwereys M. Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites. Biochemistry. 1992 Jun 23;31(24):5449–5458. doi: 10.1021/bi00139a005. [DOI] [PubMed] [Google Scholar]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Loviny-Anderton T., Shaw P. C., Shin M. K., Hartley B. S. D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Gene cloning, sequence and expression. Biochem J. 1991 Jul 1;277(Pt 1):263–271. doi: 10.1042/bj2770263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  12. Quax W. J., Mrabet N. T., Luiten R. G., Schuurhuizen P. W., Stanssens P., Lasters I. Enhancing the thermostability of glucose isomerase by protein engineering. Biotechnology (N Y) 1991 Aug;9(8):738–742. doi: 10.1038/nbt0891-738. [DOI] [PubMed] [Google Scholar]
  13. Rangarajan M., Asboth B., Hartley B. S. Stability of Arthrobacter D-xylose isomerase to denaturants and heat. Biochem J. 1992 Aug 1;285(Pt 3):889–898. doi: 10.1042/bj2850889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rangarajan M., Hartley B. S. Mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase. Biochem J. 1992 Apr 1;283(Pt 1):223–233. doi: 10.1042/bj2830223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rey F., Jenkins J., Janin J., Lasters I., Alard P., Claessens M., Matthyssens G., Wodak S. Structural analysis of the 2.8 A model of Xylose isomerase from Actinoplanes missouriensis. Proteins. 1988;4(3):165–172. doi: 10.1002/prot.340040303. [DOI] [PubMed] [Google Scholar]
  16. Schmidt-Ullrich R., Thompson W. S., Wallach D. F. Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40. Proc Natl Acad Sci U S A. 1977 Feb;74(2):643–647. doi: 10.1073/pnas.74.2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Siddiqui K. S., Rangarajan M., Hartley B. S., Kitmitto A., Panico M., Blench I. P., Morris H. R. Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin. Biochem J. 1993 Jan 1;289(Pt 1):201–208. doi: 10.1042/bj2890201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smart O. S., Akins J., Blow D. M. Molecular mechanics simulations of a conformational rearrangement of D-xylose in the active site of D-xylose isomerase. Proteins. 1992 Apr;13(2):100–111. doi: 10.1002/prot.340130203. [DOI] [PubMed] [Google Scholar]
  19. Smith C. A., Rangarajan M., Hartley B. S. D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Purification and properties. Biochem J. 1991 Jul 1;277(Pt 1):255–261. doi: 10.1042/bj2770255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Suggs S. V., Wallace R. B., Hirose T., Kawashima E. H., Itakura K. Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6613–6617. doi: 10.1073/pnas.78.11.6613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thornton J. M., Gardner S. P. Protein motifs and data-base searching. Trends Biochem Sci. 1989 Jul;14(7):300–304. doi: 10.1016/0968-0004(89)90069-8. [DOI] [PubMed] [Google Scholar]
  22. Whitlow M., Howard A. J., Finzel B. C., Poulos T. L., Winborne E., Gilliland G. L. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose. Proteins. 1991;9(3):153–173. doi: 10.1002/prot.340090302. [DOI] [PubMed] [Google Scholar]
  23. Wong H. C., Ting Y., Lin H. C., Reichert F., Myambo K., Watt K. W., Toy P. L., Drummond R. J. Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus. J Bacteriol. 1991 Nov;173(21):6849–6858. doi: 10.1128/jb.173.21.6849-6858.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. van Tilbeurgh H., Jenkins J., Chiadmi M., Janin J., Wodak S. J., Mrabet N. T., Lambeir A. M. Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis. Biochemistry. 1992 Jun 23;31(24):5467–5471. doi: 10.1021/bi00139a007. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES