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Journal of Bacteriology, August 2002, p. 4442-4448, Vol. 184, No. 16
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.16.4442-4448.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification, Overproduction, and Partial Characterization of ß-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway{dagger}

Joseph W. Scott and Madeline E. Rasche*

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

Received 28 September 2001/ Accepted 22 May 2002

Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme ß-ribofuranosylaminobenzene 5'-phosphate synthase (ß-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form ß-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding ß-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of ß-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative ß-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the ß-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential ß-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.

* Corresponding author. Mailing address: Microbiology and Cell Science Department, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700. Phone: (352) 392-1192. Fax: (352) 392-5922. E-mail: mrasche{at}ufl.edu. {dagger}Florida Agricultural Experiment Station Journal Series no. R08895.

Journal of Bacteriology, August 2002, p. 4442-4448, Vol. 184, No. 16
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.16.4442-4448.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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