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Journal of Bacteriology, February 1999, p. 1171-1180, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

Roopali Roy,1 Swarnalatha Mukund,1,dagger Gerrit J. Schut,1 Dianne M. Dunn,2 Robert Weiss,2 and Michael W. W. Adams1,*

Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602,1 and Department of Genetics, University of Utah, Salt Lake City, Utah 841122

Received 9 July 1998/Accepted 3 December 1998

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 µM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

* Corresponding author. Mailing address: Department of Biochemistry, Life Sciences Bldg., University of Georgia, Athens, GA 30602-7229. Phone: (706) 542-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.

dagger Present address: Monsanto, St. Louis, MO 63198.

Journal of Bacteriology, February 1999, p. 1171-1180, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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