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Journal of Bacteriology, May 2006, p. 3543-3550, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3543-3550.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Studies of the CobA-Type ATP:Co(I)rrinoid Adenosyltransferase Enzyme of Methanosarcina mazei Strain Gö1

Nicole R. Buan,{dagger} Kimberly Rehfeld, and Jorge C. Escalante-Semerena*

Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53726

Received 17 January 2006/ Accepted 28 February 2006

Although methanogenic archaea use B12 extensively as a methyl carrier for methanogenesis, little is known about B12 metabolism in these prokaryotes or any other archaea. To improve our understanding of how B12 metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobAMm here) was cloned and used to restore coenzyme B12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobAMm protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca2+ ions, but the activity was not enhanced by Mg2+ and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn2+. The CobAMm enzyme had a KmATP of 3 µM and a KmHOCbl of 1 µM. Unlike the S. enterica enzyme, CobAMm used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the ß phosphate of ATP was required for binding to the enzyme. A striking difference between CobASe and CobAMm was the use of ADP as a substrate by CobAMm, suggesting an important role for the {gamma} phosphate of ATP in binding. The results from 31P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPPi) is the reaction by-product; no cleavage of PPPi was observed, and the enzyme was only slightly inhibited by pyrophosphate (PPi). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobASe and CobAMm enzymes.

* Corresponding author. Mailing address: 144A Enzyme Institute, 1710 University Ave., Madison, WI 53726-4087. Phone: (608) 262-7379. Fax: (608) 265-7909. E-mail: escalante{at}bact.wisc.edu.

{dagger} Present address: Department of Microbiology University of Illinois-Urbana, Urbana, Ill.

Journal of Bacteriology, May 2006, p. 3543-3550, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3543-3550.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gray, M. J., Escalante-Semerena, J. C. (2009). In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1. J. Bacteriol. 191: 3842-3851 [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]  
  • Maurice, M. St., Mera, P. E., Taranto, M. P., Sesma, F., Escalante-Semerena, J. C., Rayment, I. (2007). Structural Characterization of the Active Site of the PduO-Type ATP:Co(I)rrinoid Adenosyltransferase from Lactobacillus reuteri. J. Biol. Chem. 282: 2596-2605 [Abstract] [Full Text]  


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