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. 1989 Jan;86(1):32–36. doi: 10.1073/pnas.86.1.32

Cloning and expression of the gene cluster encoding key proteins involved in acetyl-CoA synthesis in Clostridium thermoaceticum: CO dehydrogenase, the corrinoid/Fe-S protein, and methyltransferase.

D L Roberts 1, J E James-Hagstrom 1, D K Garvin 1, C M Gorst 1, J A Runquist 1, J R Baur 1, F C Haase 1, S W Ragsdale 1
PMCID: PMC286397  PMID: 2911576

Abstract

Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an approximately 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl beta-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum.

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Selected References

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

  1. Danna K. J. Determination of fragment order through partial digests and multiple enzyme digests. Methods Enzymol. 1980;65(1):449–467. doi: 10.1016/s0076-6879(80)65055-1. [DOI] [PubMed] [Google Scholar]
  2. Drake H. L., Hu S. I., Wood H. G. Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. Properties of phosphotransacetylase. J Biol Chem. 1981 Nov 10;256(21):11137–11144. [PubMed] [Google Scholar]
  3. Elliott J. I., Brewer J. M. The inactivation of yeast enolase by 2,3-butanedione. Arch Biochem Biophys. 1978 Sep;190(1):351–357. doi: 10.1016/0003-9861(78)90285-0. [DOI] [PubMed] [Google Scholar]
  4. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  5. Hu S. I., Pezacka E., Wood H. G. Acetate synthesis from carbon monoxide by Clostridium thermoaceticum. Purification of the corrinoid protein. J Biol Chem. 1984 Jul 25;259(14):8892–8897. [PubMed] [Google Scholar]
  6. 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]
  7. Ljungdahl L. G., Andreesen J. R. Formate dehydrogenase, a selenium--tungsten enzyme from Clostridium thermoaceticum. Methods Enzymol. 1978;53:360–372. doi: 10.1016/s0076-6879(78)53042-5. [DOI] [PubMed] [Google Scholar]
  8. Ljungdahl L. G. Physiology of thermophilic bacteria. Adv Microb Physiol. 1979;19:149–243. doi: 10.1016/s0065-2911(08)60199-x. [DOI] [PubMed] [Google Scholar]
  9. Ljungdahl L. G. The autotrophic pathway of acetate synthesis in acetogenic bacteria. Annu Rev Microbiol. 1986;40:415–450. doi: 10.1146/annurev.mi.40.100186.002215. [DOI] [PubMed] [Google Scholar]
  10. Lovell C. R., Przybyla A., Ljungdahl L. G. Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase. Arch Microbiol. 1988;149(4):280–285. doi: 10.1007/BF00411642. [DOI] [PubMed] [Google Scholar]
  11. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  12. Pezacka E., Wood H. G. Acetyl-CoA pathway of autotrophic growth. Identification of the methyl-binding site of the CO dehydrogenase. J Biol Chem. 1988 Nov 5;263(31):16000–16006. [PubMed] [Google Scholar]
  13. Ragsdale S. W., Clark J. E., Ljungdahl L. G., Lundie L. L., Drake H. L. Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein. J Biol Chem. 1983 Feb 25;258(4):2364–2369. [PubMed] [Google Scholar]
  14. Ragsdale S. W., Lindahl P. A., Münck E. Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum. J Biol Chem. 1987 Oct 15;262(29):14289–14297. [PubMed] [Google Scholar]
  15. Ragsdale S. W., Ljungdahl L. G., DerVartanian D. V. 13C and 61Ni isotope substitutions confirm the presence of a nickel (III)-carbon species in acetogenic CO dehydrogenases. Biochem Biophys Res Commun. 1983 Sep 15;115(2):658–665. doi: 10.1016/s0006-291x(83)80195-8. [DOI] [PubMed] [Google Scholar]
  16. Ragsdale S. W., Ljungdahl L. G., DerVartanian D. V. Isolation of carbon monoxide dehydrogenase from Acetobacterium woodii and comparison of its properties with those of the Clostridium thermoaceticum enzyme. J Bacteriol. 1983 Sep;155(3):1224–1237. doi: 10.1128/jb.155.3.1224-1237.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ragsdale S. W., Wood H. G. Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis. J Biol Chem. 1985 Apr 10;260(7):3970–3977. [PubMed] [Google Scholar]
  18. Ragsdale S. W., Wood H. G., Antholine W. E. Evidence that an iron-nickel-carbon complex is formed by reaction of CO with the CO dehydrogenase from Clostridium thermoaceticum. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6811–6814. doi: 10.1073/pnas.82.20.6811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shanmugasundaram T., Ragsdale S. W., Wood H. G. Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii. Biofactors. 1988 Jul;1(2):147–152. [PubMed] [Google Scholar]
  22. Slater G. G. Stable pattern formation and determination of molecular size by pore-limit electrophoresis. Anal Chem. 1969 Jul;41(8):1039–1041. doi: 10.1021/ac60277a003. [DOI] [PubMed] [Google Scholar]
  23. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  24. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  25. Wood H. G., Ragsdale S. W., Pezacka E. A new pathway of autotrophic growth utilizing carbon monoxide or carbon dioxide and hydrogen. Biochem Int. 1986 Mar;12(3):421–440. [PubMed] [Google Scholar]
  26. Yang H. M., Reisfeld R. A. Pharmacokinetics and mechanism of action of a doxorubicin-monoclonal antibody 9.2.27 conjugate directed to a human melanoma proteoglycan. J Natl Cancer Inst. 1988 Sep 21;80(14):1154–1159. doi: 10.1093/jnci/80.14.1154. [DOI] [PubMed] [Google Scholar]

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