It is proposed to isolate a library of genomic DNA from the archaebacter Methanococcus jannaschii. The organism is an extreme thermo- and barophile, its natural habitat being in the vicinity of a deep-sea hydrothermal vent at a pressure of 260 atm. (Methanococcus jannaschii is the most thermophilic organism from which DNA has been isolated with a laboratory growth optimum of 92oC). The DNA from this extraordinary microorganism will be cloned into Escherichia coli using standard molecular biology procedures. The genomic library will be screened for the gene encoding a hydrogenase enzyme. Expression of the hydrogenase gene, using an approach which has already proved successful in cloning methanogenic bacterial genes, will enable not only the investigation of the processes controlling extreme thermostability and barophilicity, but also the production of a potentially valuable commercial enzyme. Using the approaches of site-directed and random mutagenesis a family of similar enzymes with differing responses to temperature and pressure will be generated. This series of closely related mutant enzymes will be far easier to compare than a set of related hydrogenases isolated from meso- and thermophiles. Since the enzyme is extremely thermostable, it should be possible to delineate some of the structure-function relationships which give rise to marked stability. Of particular interest is the response of the hydrogenase to increased pressure (especially since there could be a physiological role for such a response in deep sea organisms). The pressure regulation of this enzyme will be investigated in detail. Some enzymes are activated or inactivated by large pressure changes, but, this hydrogenase is the first enzyme which triples activity over a relatively small change in pressure from 7.5 to 260 atm. The kinetic results of the proposed research will be related to the structure of the enzyme. In addition, enough enzyme will be purified to enable an early attempt at crystallization.
