Summary auto-generated
Researchers characterized the [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum. The enzyme is a tetrameric α₂β₂ complex with 50 kDa and 25 kDa subunits. Key properties include specific activity of 77 pmol/min per mg protein, pH optimum of 7.5, thermal stability (T50 ~70°C), and remarkable oxygen resistance, retaining full activity after 24 hours of air exposure. Using N-terminal protein sequences, researchers isolated and sequenced the corresponding genes (hydS and hydL). The enzyme is a typical [Ni-Fe] hydrogenase, most closely related to Class IV enzymes from other organisms. When heterologously expressed in Escherichia coli, the recombinant enzyme accumulated at high levels in soluble form but remained inactive. Analysis revealed the recombinant large subunit lacked proper C-terminal post-translational processing, explaining the inactivity. The enzyme demonstrates the structural and biochemical features characteristic of [Ni-Fe] hydrogenases despite previous misclassification as a [Fe]-only enzyme.
Key findings
- The A. flavidum hydrogenase is an α₂β₂ tetramer with high specific activity (77 pmol/min per mg) and unusual oxygen stability, retaining activity after 24 hours air exposure
- Molecular characterization revealed typical [Ni-Fe] enzyme structure with genes (hydS, hydL) showing highest homology to Class IV [Ni-Fe] hydrogenases from Alcaligenes eutrophus
- Heterologous expression in E. coli produced high levels of soluble protein lacking catalytic activity due to failure of C-terminal post-translational processing of the large subunit
- The enzyme has a T50 of ~70°C and pH optimum of 7.5, making it well-adapted to thermophilic conditions in Acetomicrobium flavidum
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Abstract
Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.