Swarnalatha Mukund

Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase

The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.

Influence of tungsten on metabolic patterns in Pyrococcus furiosus, a hyperthermophilic archaeon

Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally around 100 °C. It can be cultured in an artificial seawater-based medium with either peptides or maltose as the carbon source. Significant stimulation of cell yields were observed when trace levels of tungsten (as tungstate) were added to an energy-limited chemostat culture of P. furiosus when maltose is present, but not when peptides were the sole carbon source. The addition of tungsten also led to dramatic increases in the specific activities within cell-free extracts of aldehyde ferredoxin oxidoreductase (AOR), which is a tungsten-iron-sulfur protein. Moreover, the addition of tungsten to cells growing in maltose/peptide medium dramatically reduced the specific activity of intracellular proteases, suggesting a preference for the utilization of maltose over peptides as the carbon and energy source in the presence of tungsten.

Two-dimensional gel electrophoresis mapping of proteins isolated from the hyperthermophile Pyrococcus furiosus

Abstract Two-dimensional gel electrophoresis (2DE) in polyacrylamide was used to map the proteins in lysates of the archaeon (formerly archaebacterium) Pyrococcus furiosus and to analyze enzymes purified from P. furiosus. The location of the enzymes in the 2DE maps was determined by comigration of lysate proteins with purified enzymes. A 2DE map of P. furiosus proteins with some identifications was produced, which will be useful for future studies of protein expression in this organism. In addition, the usefulness of 2DE for evaluating the purity of enzyme preparations and for characterizing their subunit structure under denaturing conditions was investigated.