Presidential Young Investigator Award

本研究拟从jannaschii甲烷球菌中分离出一个基因组DNA文库。这种生物是一种极端的嗜热嗜水动物，它的自然栖息地是在一个压力为260大气压的深海热液喷口附近。（jannaschii甲烷球菌是最嗜热的生物，其DNA已被分离出来，实验室生长最佳温度为92℃）。这种特殊微生物的DNA将通过标准的分子生物学程序克隆到大肠杆菌中。基因组文库将筛选编码氢化酶的基因。氢化酶基因的表达，使用一种已经被证明在克隆产甲烷细菌基因上成功的方法，不仅可以研究控制极端热稳定性和嗜压性的过程，而且还可以生产一种潜在有价值的商业酶。利用定点诱变和随机诱变的方法，将产生一系列对温度和压力有不同反应的类似酶。这一系列密切相关的突变酶将比从中嗜热菌和嗜热菌中分离出的一组相关的氢化酶更容易比较。由于酶具有极强的热稳定性，因此应该有可能描绘出一些结构-功能关系，这些关系产生了显著的稳定性。特别令人感兴趣的是氢化酶对压力增加的反应（特别是因为这种反应在深海生物中可能具有生理作用）。我们将详细研究这种酶的压力调节。有些酶在较大的压力变化下被激活或灭活，但是，这种氢化酶是第一个在压力从7.5到260大气压的相对较小变化下活性增加三倍的酶。本研究的动力学结果将与酶的结构有关。此外，足够的酶将被纯化，使早期的结晶尝试。

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.