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. 1987 Jun 15;244(3):585–590. doi: 10.1042/bj2440585

Purification and some properties of a novel heat-stable cis-toluene dihydrodiol dehydrogenase.

H D Simpson 1, J Green 1, H Dalton 1
PMCID: PMC1148036  PMID: 3446178

Abstract

cis-Toluene dihydrodiol dehydrogenase was purified 200-fold from cells of a thermotolerant Bacillus species grown with toluene as the sole source of carbon and energy. The purified enzyme preparation was remarkably heat-stable and exhibited a half-life of 100 min at 80 degrees C, the temperature optimum. The activation energy of the reaction was 36 kJ.mol-1. Isoelectric focusing indicated that the pI of the native enzyme was 6.4 and that of the denatured enzyme 6.5. Although the pH optimum was 9.8, the enzyme was most stable at pH 8. The Mr of the enzyme was approx. 172,000 as determined by gel filtration and 166,000 by polyacrylamide-gel electrophoresis. The enzyme was composed of six apparently identical subunits with Mr values of 29,500. Kinetic analysis revealed that the Km for cis-toluene dihydrodiol was 92 microM and for NAD+ was 80 microM. The apparent Km values for cis-benzene dihydrodiol and cis-naphthalene dihydrodiol were 330 microM and 51 microM respectively. The enzyme was inhibited by mercurials but was unaffected by metal-ion chelators. Steady-state kinetics and product-inhibition patterns suggested that the enzyme mechanism was ordered Bi Bi.

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Selected References

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

  1. Amelunxen R. E. Crystallization of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. Biochim Biophys Acta. 1966 Aug 10;122(2):175–181. doi: 10.1016/0926-6593(66)90059-2. [DOI] [PubMed] [Google Scholar]
  2. Axcell B. C., Geary P. J. The metabolism of benzene by bacteria. Purification and some properties of the enzyme cis-1,2-dihydroxycyclohexa-3,5-diene (nicotinamide adenine dinucleotide) oxidoreductase (cis-benzene glycol dehydrogenase). Biochem J. 1973 Dec;136(4):927–934. doi: 10.1042/bj1360927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eberspächer J., Lingens F. Reinigung und Eigenschaften von zwei Chloridazondihydrodiol-Dehydrogenasen aus Chloridazon-abbauenden Bakterien. Hoppe Seylers Z Physiol Chem. 1978 Oct;359(10):1323–1334. [PubMed] [Google Scholar]
  4. Greene R. L., Street G. B. Conducting organic materials. Science. 1984 Nov 9;226(4675):651–656. doi: 10.1126/science.226.4675.651. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Matsunaga A., Noso Y. Conformational change with temperature and thermostability of glutamine synthetase from Bacillus stearothermophilus. Biochim Biophys Acta. 1974 Sep 13;365(1):208–211. doi: 10.1016/0005-2795(74)90265-7. [DOI] [PubMed] [Google Scholar]
  7. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  8. Oesch F., Daly J. Conversion of naphthalene to trans-naphthalene dihydrodiol: evidence for the presence of a coupled aryl monooxygenase-epoxide hydrase system in hepatic microsomes. Biochem Biophys Res Commun. 1972 Feb 25;46(4):1713–1720. doi: 10.1016/0006-291x(72)90807-8. [DOI] [PubMed] [Google Scholar]
  9. Patel T. R., Gibson D. T. Purification and propeties of (plus)-cis-naphthalene dihydrodiol dehydrogenase of Pseudomonas putida. J Bacteriol. 1974 Sep;119(3):879–888. doi: 10.1128/jb.119.3.879-888.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Reiner A. M. Metabolism of aromatic compounds in bacteria. Purification and properties of the catechol-forming enzyme, 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (NAD + ) oxidoreductase (decarboxylating). J Biol Chem. 1972 Aug 25;247(16):4960–4965. [PubMed] [Google Scholar]
  11. Rogers J. E., Gibson D. T. Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida. J Bacteriol. 1977 Jun;130(3):1117–1124. doi: 10.1128/jb.130.3.1117-1124.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Singleton R., Jr, Amelunxen R. E. Proteins from thermophilic microorganisms. Bacteriol Rev. 1973 Sep;37(3):320–342. doi: 10.1128/br.37.3.320-342.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. VISHNIAC W., SANTER M. The thiobacilli. Bacteriol Rev. 1957 Sep;21(3):195–213. doi: 10.1128/br.21.3.195-213.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yeh W. K., Gibson D. T., Liu T. N. Toluene dioxygenase: a multicomponent enzyme system. Biochem Biophys Res Commun. 1977 Sep 9;78(1):401–410. doi: 10.1016/0006-291x(77)91268-2. [DOI] [PubMed] [Google Scholar]

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