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 Caccamo, M. A. Articles by Rasche, M. E. Search for Related Content PubMed PubMed Citation Articles by Caccamo, M. A. Articles by Rasche, M. E.

 Previous Article  |  Next Article 

Journal of Bacteriology, April 2004, p. 2068-2073, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.2068-2073.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of a Dihydromethanopterin Reductase Involved in Tetrahydromethanopterin Biosynthesis in Methylobacterium extorquens AM1

Marco A. Caccamo, Courtney S. Malone, and Madeline E. Rasche^*

Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611-0700

Received 19 June 2003/ Accepted 11 December 2003

During growth on one-carbon (C₁) compounds, the aerobic -proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H₄MPT) derivative dephospho-H₄MPT as a C₁ carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H₄MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H₄MPT biosynthesis is the reduction of dihydromethanopterin (H₂MPT) to H₄MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H₂MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H₂MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H₂MPT with a specific activity of 2.8 µmol of NADPH oxidized per min per mg of protein at 30°C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H₂MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F₄₂₀, used, respectively, in bacteria and methanogenic archaea.

Florida Agricultural Experiment Station Journal Series number R-09922.

Present address: College of Osteopathic Medicine and Surgery, Des Moines University, Des Moines, IA 50312.

This article has been cited by other articles:

• Kalyuzhnaya, M. G., Korotkova, N., Crowther, G., Marx, C. J., Lidstrom, M. E., Chistoserdova, L. (2005). Analysis of Gene Islands Involved in Methanopterin-Linked C1 Transfer Reactions Reveals New Functions and Provides Evolutionary Insights. J. Bacteriol. 187: 4607-4614 [Abstract] [Full Text]  
• Chistoserdova, L., Rasche, M. E., Lidstrom, M. E. (2005). Novel Dephosphotetrahydromethanopterin Biosynthesis Genes Discovered via Mutagenesis in Methylobacterium extorquens AM1. J. Bacteriol. 187: 2508-2512 [Abstract] [Full Text]