homologous expression of a subcomplex of pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase复形的同源表达海床furiosus氢化酶,与丙酮酸铁氧还蛋白氧化还原酶.pdfVIP

Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase . .¤a ¤b R. Christopher Hopkins , Junsong Sun , Francis E. Jenney, Jr. , Sanjeev K. Chandrayan, Patrick M. McTernan, Michael W. W. Adams* Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America Abstract Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100uC. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP- dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found