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2003 Annual Science Report

Pennsylvania State University Reporting  |  JUL 2002 – JUN 2003

Enzymes of Ancient Metabolic Pathways

4 Institutions
3 Teams
0 Publications
0 Field Sites
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Project Progress

The protein (AfpA, archaeoflavoprotein) encoded by AF1518 in the genome of Archeoglobus fulgidus was produced in Escherichia coli and characterized. AfpA was found to be a homodimer with a native molecular mass of 43 kDa containing two noncovalently bound FMN. Cell extract of A. fulgidus catalyzed the CO-dependent reduction of AfpA that was stimulated by the addition of ferredoxin. Ferredoxin was found to be a direct electron donor to AfpA, whereas rubredoxin was unable to substitute. Neither NADH + H+ nor NADPH + H+ was an electron donor. Ferricyanide, 2,6-dichlorophenolindophenol, several quinones, ferric citrate, bovine cytochrome c, and O2 accepted electrons from reduced AfpA (AfpAred), whereas coenzyme F420 did not. The FMN in AfpAred was fully oxidized by all the quinones tested with a 1:1 ratio. No evidence for a flavin semiquinone was observed in absorbence spectra of AfpAred during oxidation by the electron acceptors. The rate of cytochrome c reduction was enhanced in the presence of O2, suggesting that superoxide is a product of the interaction of AfpAred with O2. Sequence analyses of AfpA homologs identified a strictly conserved FMN binding motif unique to the homo-oligomeric flavin-containing cysteine decarboxylase (HFCD) superfamily. A search of the databases suggests that AfpA is the prototype of a previously unrecognized family unique to the Archaea domain for which the name archaeoflavoprotein is proposed. The genomes of several diverse Archaea contained afpA homologs clustered with open reading frames annotated as putative homologs of genes encoding proteins involved in the oxidative stress response of anaerobes. In summary, the results demonstrate that AF1518 encodes the prototype of a previously unrecognized family of FMN-containing flavoproteins identified only in A. fulgidus and methane-producing species from the Archaea domain.

    James Ferry James Ferry
    Project Investigator
    Yanhuai Ding

    Christie Brosius
    Doctoral Student

    Francisco Cruz
    Doctoral Student

    Caleb Bell III
    Undergraduate Student

    Objective 3.3
    Origins of energy transduction

    Objective 4.1
    Earth's early biosphere

    Objective 5.1
    Environment-dependent, molecular evolution in microorganisms

    Objective 5.2
    Co-evolution of microbial communities

    Objective 5.3
    Biochemical adaptation to extreme environments

    Objective 6.1
    Environmental changes and the cycling of elements by the biota, communities, and ecosystems

    Objective 6.2
    Adaptation and evolution of life beyond Earth