Molecular Characterization of the [Ni-Fe] Hydrogenase from the Thermophilic Bacterium Acetomicrobium flavidum
Elisabetta FRANCHI, Claudio TOSI, Francesco RODRIGUEZ, Alessandro SELVAGGI, and Paola PEDRONI*
Eniricerche S.p.A., Environmental Technologies, Via F. Maritano 26, San Donato Milanese 20097, Milan, Italy
The progressive depletion of fossil fuels and the pollution problems associated to their combustion will make the identification of clean and renewable alternatives necessary in a near future. Hydrogen is considered among the environmentally friendly energy sources and its biological production might represent an attractive possibility from the economical point of view. Hydrogenases are the enzymes directly involved in the hydrogen metabolism of many microorganisms since enable them to use hydrogen either as an energy source (H2 uptake) or as an electron sink (H2 evolution), according to the reaction H2 ←→ 2H+＋2e-.
We work as part of a long-term project supported by the Japanese New Energy and Industrial Technology Development Organization (NEDO) also providing the biochemical and the genetic characterization of different hydrogenases, as a fundamental prerequisite to optimize the hydrogen evolution capacity of microorganisms.
Acetomicrobium flavidum is an anaerobic thermophilic bacterium optimally growing at 60℃ which ferments 1 mole of glucose to 2 moles of acetate and CO2 and 4 moles of H2. Its H2-evolving hydrogenase is one of the enzymes we focused our attention on.
The biochemical characterization evidenced that A. flavidum hydrogenase is an heterodimeric enzyme composed of two different subunits (molecular mass of 50kDa and 25 kDa, respectively) arranged in an α2β2 tetramer exhibiting particular features attractive for applicative purposes (i.e. catalytic efficiency, thermostability and resistance to oxygen inactivation).
The characterization of the corresponding gene cluster showed conserved amino acid motifs in the small and large subunit sequences, indicating that the enzyme is a [Ni-Fe] hydrogenase of Class IV, and the presence of additional ORFs located downstream from the structural hydSL genes. One of these, named hydD, encodes a polypeptide homologous to hydrogenase-related proteins indicated as the proteases which cleave the large subunits at their C-terminal ends.
The expression in E. coli of the hydSL structural genes gave rise to a soluble but inactive enzyme and the analysis of the recombinant large subunit showed that it was expressed as a precursor. Our results therefore confirm that the large subunit proteolytic processing represents one of the fundamental events in the formation of active [Ni-Fe] hydrogenases, suggest a strict cleavage specificity, since none of the three (or four) homologous proteases present in E. coli is able to process the recombinant subunit and indicate that the co-expression of ancillary genes is required to obtain a functional enzyme.
Assuming that the specific protease is encoded by the hydD gene, we are using A. flavidum hydrogenase as a model system to elucidate this posttranslational maturation step. We are verifying in vivo the formation of processed A. flavidum large subunit as mediated by the specific protease HydD, by co-expressing in E. coli hydSL genes togheter with hydD.
