This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thomas, M. G. Articles by Escalante-Semerena, J. C. Search for Related Content PubMed PubMed Citation Articles by Thomas, M. G. Articles by Escalante-Semerena, J. C.

Journal of Bacteriology, August 2000, p. 4227-4233, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of an Alternative Nucleoside Triphosphate: 5'-Deoxyadenosylcobinamide Phosphate Nucleotidyltransferase in Methanobacterium thermoautotrophicum H

Michael G. Thomas and Jorge C. Escalante-Semerena^*

Department of Bacteriology, University of WisconsinMadison, Madison, Wisconsin

Received 15 March 2000/Accepted 4 May 2000

Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.

---

^* Corresponding author. Mailing address: Department of Bacteriology, University of WisconsinMadison, 1550 Linden Dr., Madison, WI 53706-1567. Phone: (608) 262-7379. Fax: (608) 262-9865. E-mail: jcescala{at}facstaff.wisc.edu.

---

Journal of Bacteriology, August 2000, p. 4227-4233, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Lawrence, A. D., Deery, E., McLean, K. J., Munro, A. W., Pickersgill, R. W., Rigby, S. E. J., Warren, M. J. (2008). Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis. J. Biol. Chem. 283: 10813-10821 [Abstract] [Full Text]  
• Zayas, C. L., Claas, K., Escalante-Semerena, J. C. (2007). The CbiB Protein of Salmonella enterica Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway. J. Bacteriol. 189: 7697-7708 [Abstract] [Full Text]  
• Otte, M. M., Woodson, J. D., Escalante-Semerena, J. C. (2007). The Thiamine Kinase (YcfN) Enzyme Plays a Minor but Significant Role in Cobinamide Salvaging in Salmonella enterica. J. Bacteriol. 189: 7310-7315 [Abstract] [Full Text]  
• Escalante-Semerena, J. C. (2007). Conversion of Cobinamide into Adenosylcobamide in Bacteria and Archaea. J. Bacteriol. 189: 4555-4560 [Full Text]  
• Zayas, C. L., Escalante-Semerena, J. C. (2007). Reassessment of the Late Steps of Coenzyme B12 Synthesis in Salmonella enterica: Evidence that Dephosphorylation of Adenosylcobalamin-5'-Phosphate by the CobC Phosphatase Is the Last Step of the Pathway. J. Bacteriol. 189: 2210-2218 [Abstract] [Full Text]  
• Woodson, J. D., Escalante-Semerena, J. C. (2006). The cbiS Gene of the Archaeon Methanopyrus kandleri AV19 Encodes a Bifunctional Enzyme with Adenosylcobinamide Amidohydrolase and {alpha}-Ribazole-Phosphate Phosphatase Activities.. J. Bacteriol. 188: 4227-4235 [Abstract] [Full Text]  
• Buan, N. R., Rehfeld, K., Escalante-Semerena, J. C. (2006). Studies of the CobA-Type ATP:Co(I)rrinoid Adenosyltransferase Enzyme of Methanosarcina mazei Strain Go1.. J. Bacteriol. 188: 3543-3550 [Abstract] [Full Text]  
• Zayas, C. L., Woodson, J. D., Escalante-Semerena, J. C. (2006). The cobZ Gene of Methanosarcina mazei Go1 Encodes the Nonorthologous Replacement of the {alpha}-Ribazole-5'-Phosphate Phosphatase (CobC) Enzyme of Salmonella enterica.. J. Bacteriol. 188: 2740-2743 [Abstract] [Full Text]  
• Maggio-Hall, L. A., Claas, K. R., Escalante-Semerena, J. C. (2004). The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea. Microbiology 150: 1385-1395 [Abstract] [Full Text]  
• Woodson, J. D., Escalante-Semerena, J. C. (2004). CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea. Proc. Natl. Acad. Sci. USA 101: 3591-3596 [Abstract] [Full Text]  
• Woodson, J. D., Zayas, C. L., Escalante-Semerena, J. C. (2003). A New Pathway for Salvaging the Coenzyme B12 Precursor Cobinamide in Archaea Requires Cobinamide-Phosphate Synthase (CbiB) Enzyme Activity. J. Bacteriol. 185: 7193-7201 [Abstract] [Full Text]  
• Rodionov, D. A., Vitreschak, A. G., Mironov, A. A., Gelfand, M. S. (2003). Comparative Genomics of the Vitamin B12 Metabolism and Regulation in Prokaryotes. J. Biol. Chem. 278: 41148-41159 [Abstract] [Full Text]  
• Woodson, J. D., Peck, R. F., Krebs, M. P., Escalante-Semerena, J. C. (2003). The cobY Gene of the Archaeon Halobacterium sp. Strain NRC-1 Is Required for De Novo Cobamide Synthesis. J. Bacteriol. 185: 311-316 [Abstract] [Full Text]  
• Roper, J. M., Raux, E., Brindley, A. A., Schubert, H. L., Gharbia, S. E., Shah, H. N., Warren, M. J. (2000). The Enigma of Cobalamin (Vitamin B12) Biosynthesis in Porphyromonas gingivalis. IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL CORRIN PATHWAY. J. Biol. Chem. 275: 40316-40323 [Abstract] [Full Text]  
• Fonseca, M. V., Escalante-Semerena, J. C. (2001). An in Vitro Reducing System for the Enzymic Conversion of Cobalamin to Adenosylcobalamin. J. Biol. Chem. 276: 32101-32108 [Abstract] [Full Text]