1887

Abstract

Methanol oxidation in appears to be organized in a way similar to that in other Gram-negative methylotrophs, involving a quinoprotein methanol dehydrogenase (MDH; EC 1.1.99.8) and two soluble cytochromes. The MDH is composed of two different subunits, most probably arranged in an αβ structure. The two cytochromes are of the -type and differ in size (molecular mass 19.5 and 12.5 kDa) and isoelectric point (pI 4.6 and 9.2). The one with the lowest isoelectric point, commonly designated as cytochrome , is able to oxidize reduced MDH. Taking advantage of the ability of , a restricted facultative methylotroph, to utilize fructose as a growth substrate, mutants impaired in methanol utilization were isolated after application of optimal concentrations of ethylmethane sulphonate. Three classes of methanol oxidation mutants were obtained. Class I mutants were affected in a global regulation of the synthesis of both apo-methanol-dehydrogenase and cytochrome as well as PQQ (pyrroloquinoline quinone). Class II mutants did not produce an active MDH, but instead a comparable amount of a 65 kDa protein was found in the cell-free extract upon SDS-PAGE. This mutant protein was purified and compared to wild-type MDH. It was located in the periplasm, but unlike MDH it was composed of only two identical large subunits. Each of these subunits was able to bind one molecule of PQQ. An antiserum raised against wild-type MDH did not react with the mutant protein. Conversely, an antiserum raised against mutant protein weakly cross-reacted with wild-type MDH, suggesting that the presence of the β-subunit in MDH dramatically changes its immunochemical behaviour. Three of the class II mutants did not produce PQQ. In the presence of PQQ, partial revertants able to grow on methanol medium were obtained (class III). Class III mutants produced a stable apo-MDH consisting of α- and β-subunits and showing a normal reaction with the anti-MDH serum. Although this apo-MDH can bind PQQ, enzymic activity was not restored . This suggests that, in addition to apo-MDH and PQQ, other factors are required for the assembly of an enzymically active MDH.

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1992-10-01
2022-01-17
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References

  1. Adachi O., , Matsushita K., , Shinagawa E., & Ameyama M. 1990a; Calcium in quinoprotein methanol dehydrogenase can be replaced by strontium. Agricultural and Biological Chemistry 54:2833–2837
    [Google Scholar]
  2. Adachi O., , Matsushita K., , Shinagawa E., & Ameyama M. 1990b; Purification and properties of methanol dehydrogenase and alde-hyde dehydrogenase from Methylobacillus glycogenes . Agricultural and Biological Chemistry 54:3123–3129
    [Google Scholar]
  3. Adelberg E. A., , Mandel M., & Chein Ching Chen G. 1965; Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitroso-guanidine in Escherichia coli K 12. Biochemical and Biophysical Research communications 18:788–795
    [Google Scholar]
  4. Alefounder P. K., & Ferguson S. J. 1981; A periplasmic location for methanol dehydrogenase from Paracoccus denitrificans: implications for proton pumping by cytochrome aa 3 . Biochemical and Biophysical Research Communications 98:778–784
    [Google Scholar]
  5. Allen N. L., & Hanson R. S. 1985; Construction of broad-host-range cosmid Cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. Journal of Bacteriology 161:955–962
    [Google Scholar]
  6. Anderson D. J., & Lidstrom M. E. 1988; The moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AMI. Journal of Bacteriology 170:2254–2262
    [Google Scholar]
  7. Anthony C. 1986; Bacterial oxidation of methane and methanol. Advances in Microbial Physiology 27:113–210
    [Google Scholar]
  8. Anthony C. 1992; The structure of bacterial quinoproteins. International Journal of Biochemistry 24:29–39
    [Google Scholar]
  9. Biville F., , Mazodier P., , Gasser F., , Van Kleef M.A.G., & Duine J. A. 1988; Physiological properties of a pyrroloquinoline-quinone mutant of Methylobacterium organophilum . FEMS Microbiology Letters 52:53–58
    [Google Scholar]
  10. Biville F., , Turlin E., & Gasser F. 1989; Cloning and genetic analysis of six pyrroloquinoline quinone biosynthesis genes in Methylobacterium organophilium DSM 760. Journal of General Microbiology 135:2917–2929
    [Google Scholar]
  11. Brockman R. W., & Heppel L. A. 1968; On the localization of alkaline phosphatase and cyclic phosphodiesterase in Escherichia coli . Biochemical Journal 7:2554–2562
    [Google Scholar]
  12. Cross A. R., & Anthony C. 1980; The purification and properties of the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus . Biochemical Journal 192:421–427
    [Google Scholar]
  13. Dawson A., & Goodwin P. M. 1990; Investigation of mutants of Methylophilus methylotrophus which are defective in methanol oxidation. Journal of General Microbiology 136:1373–1380
    [Google Scholar]
  14. Dekker R. H., , Duine J. A., , Frank Jzn J., , Verwiel E. P., & Westerling J. 1982; Covalent addition of H2O, enzyme sub-strates and activators to pyrroloquinoline quinone, the coenzyme of quinoproteins. European Journal of Biochemistry 125:69–73
    [Google Scholar]
  15. Dijkstra M., , Frank J., , Wielink J.E., & Duine J. A. 1988; The soluble cytochromes c of methanol-grown Hyphomicrobium X. Biochemical Journal 251:464–474
    [Google Scholar]
  16. Dijkstra M., , Frank Jzn, , J., & Duine J. A. 1989; Studies on electron transfer to cytochrome cL, both purified from Hyphomicro-bium X. Biochemical Journal 257:87–94
    [Google Scholar]
  17. Dokter P., , Frank Jzn, , J., & Duine J. A. 1986; Purification and characterization of quinoprotein glucose dehydrogenase from Acine-tobacter calcoaceticus LMD 79. 41. Biochemical Journal 239:163–167
    [Google Scholar]
  18. Frank K., & Duine J. A. 1990; Cytochrome cL and cytochrome c H from Hyphomicrobium X. Methods in Enzymology 188:303–308
    [Google Scholar]
  19. Frank J., , Dijkstra M., , Balny C., , Verwiel P. E. J., & Duine J. A. 1989; Methanol dehydrogenase: mechanism of action. Antonie van Leeuwenhoek 56:25–34
    [Google Scholar]
  20. Harms N., & Van Spanning R. J. M. 1991; C1 metabolism in Paracoccus denitrificans. Genetics of Paracoccus denitrificans . Journal of Bioenergetics and Biomembranes 23:187–210
    [Google Scholar]
  21. Harms N., , De Vries G. E., , Maurer K., , Veltkamp E., & Stouthamer A. H. 1985; Isolation and characterization of Paracoccus denitrificans mutants with defects in the metabolism of one-carbon compounds. Journal of Bacteriology 164:1064–1070
    [Google Scholar]
  22. Van Iersel J., , Frank J., & Duine J. A. 1985; Determination of absorption coefficients of purified proteins by conventional ultra-violet spectrophotometry and chromatography combined with multi-wavelength detection. Analytical Biochemistry 151:196–204
    [Google Scholar]
  23. Janvier M., & Gasser F. 1987; Purification and properties of methanol dehydrogenase from Methylophaga marina . Biochimie 69:1169–1174
    [Google Scholar]
  24. Janvier M., , Frehel C., , Grimont F., & Gasser F. 1985; Methylophaga marina gen. nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs. International Journal of Systematic Bacteriology 35:131–139
    [Google Scholar]
  25. Jenkins O., , Byrom D., & Jones D. 1987; Methylophilus: a new genus of methanol-utilizing bacteria. International Journal of Systematic Bacteriology 35:446–448
    [Google Scholar]
  26. Johnson P. A., & Quayle J. R. 1964; Oxidation of formaldehyde, formate and methanol by methanol grown Pseudomonas AM I. Biochemical Journal 35:281–290
    [Google Scholar]
  27. Kletsova L. V., , Chibisova E. S., & Tsygankov Y. D. 1988; Mutants of the obligate methylotroph Methylobacillus fiagellatum KT defective in genes of the ribulose monophosphate cycle of formaldehyde fixation. Archives of Microbiology 149:441–446
    [Google Scholar]
  28. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  29. Lee K. E., , Stone S., , Goodwin P. M., & Holloway B. W. 1991; Characterization of transposition insertion mutants of Methylobac-terium extorquens AMI (Methylobacterium strain AMI) which are defective in methanol oxidation. Journal of General Microbiology 137:895–904
    [Google Scholar]
  30. Lidstom M. E. 1990; Genetics of carbon metabolism in methylo-trophic bacteria. FEMS Microbiology Reviews 87:431–436
    [Google Scholar]
  31. Lidstrom M. E., , Nunn D. N., , Anderson D. J. Stephens, , R. L., & Haygood M. G. 1987 Molecular biology of methanol oxidation. In Microbial Growth on C1 Compounds. Proceedings of the 5th International Symposium, pp. 246–254 Edited by van Verseveld H. W., & person-group-type="editor"> Duine J. A. Dordrecht, NL: Martinus Nijhoff;
    [Google Scholar]
  32. Machlin S. M., , Tam P. E., , Bastien C. A., & Hanson R. S. 1988; Genetic and physical analysis of Methylobacterium organophilum XX genes encoding methanol oxidation. Journal of Bacteriology 170:141–148
    [Google Scholar]
  33. Mazodier P., , Biville F., , Turlin E., & Gasser F. 1988; Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760. Journal of General Microbiology 134:2513–2524
    [Google Scholar]
  34. Meloch H.P., , Ingram J.M., & Wood W. A. 1966; 2-Keto-3-deoxy-6-phosphogluconic aldolase. Methods in Enzymology 9:520–528
    [Google Scholar]
  35. Miller J. H. 1972 Experiments in Molecular Genetics, pp. 125–139 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Nunn D. N., & Lidstrom M. E. 1986a; Isolation and complementa-tion analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AMI. Journal of Bacteriology 166:582–591
    [Google Scholar]
  37. Nunn D. N., & Lidstrom M. E. 1986b; Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1. Journal of Bacteriology 166:591–597
    [Google Scholar]
  38. Nunn D. N., & Anrhony C. 1988; The nucleotide sequence and deduced amino acid sequence of the cytochrome cL gene of Methylobacterium extorquens AM1, a novel class of c-type cyto-chrome. Biochemical Journal 256:673–676
    [Google Scholar]
  39. Nunn D. N., , Day D., & Anrhony C. 1989; The second subunit of methanol dehydrogenase of Methylobacterium extorquens AMI. Biochemical Journal 260:857–862
    [Google Scholar]
  40. O'Keefe D., & Anrhony C. 1980; The two cytochromes c in the facultative methylotroph Pseudomonas AMI. Biochemical Journal 192:411–419
    [Google Scholar]
  41. Peltre G., , Lapeyre J., & David B. 1982; Heterogeneity of grass pollen allergens (Dactylis glomerata) recognized by IgE antibodies in human patients' sera by a new nitrocellulose immunoprint technique. immunology Letters 5:127–131
    [Google Scholar]
  42. Reed L. J., & Mukherjee B. B. 1969; Alpha-Ketoglutarate dehydrogenase complex from Escherichia coli . Methods in Enzy-mology 12:55–61
    [Google Scholar]
  43. Reisfeed R. A., , Lewis H. J., & Williams D. E. 1962; Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature, London 195:281–283
    [Google Scholar]
  44. Van Spanning R.J.M., , Wansell C. W., , De Boer T., , Hazelaar M. J., , Anazawa H., , Harms N., , Oltmann M. J., & Stouthamer A. H. 1991; Isolation and characterization of the moxJ, moxG, moxl, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth. Journal of Bacteriology 173:6948–6961
    [Google Scholar]
  45. Stirling D. I., & Dalton H. 1978; Purification and properties of a NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath). Journal of General Microbiology 107:19–29
    [Google Scholar]
  46. Tatra P. K., & Goodwin P. M. 1985; Mapping of some genes involved in C-1 metabolism in the facultative methylotroph Methylobacterium sp. strain AMI (Pseudomonas AMI). Archives of Microbiology 143:169–177
    [Google Scholar]
  47. Thomas P. E., , Ryan D., & Levin W. 1976; An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical Biochemistry 15, 168–176
    [Google Scholar]
  48. Towbin H., , Staehelin T., & Gordon J. 1979; Electrophoretic transfer from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 764350–4354
    [Google Scholar]
  49. Uhlig H., , Karbaum K., & Steudel A. 1986; Acetobacter methanolicus sp. nov., an acidophilic facultatively methylotrophic bacterium. International Journal of Systematic Bacteriology 36:317–322
    [Google Scholar]
  50. Urakami T., & Komagata K. 1986; Emendation of Methylobacillus Yordy and Weaver 1977, a genus for methanol-utilizing bacteria. International Journal of Systematic Bacteriology 36:502–511
    [Google Scholar]
  51. De Vries C. E., , Koes U., & Stahl U. 1990; Physiology and genetics of methylotrophic bacteria. FEMS Mic;·obiology Reviews 15, 57–102
    [Google Scholar]
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