The relevance of being out of order: requirement of Intrinsically Disordered Regions for metalloenzyme biosynthesis

Hydrogen is recognized as an essential energy vector on the low-carbon economy that should be implemented to alleviate the huge problems derived from the massive use of fossil fuels. From a biological point of view, hydrogen is both a fuel for cell metabolism and a way to equilibrate redox balance in fermentative and photosynthetic metabolisms through proton reduction. Hydrogenases are key metalloenzymes for the bioproduction and oxidation of hydrogen with potential biotech applications, such as the generation of bio-based fuel cells. The activity of hydrogenases also improves the energy efficiency of nitrogen fixation by recycling the hydrogen evolved from nitrogenase.

The synthesis of hydrogenases involves a complex series of biochemical reactions to assemble protein subunits and metallic cofactors required for enzyme function through a mechanism still not well understood. A final step in this biosynthetic pathway is the processing of a C-terminal tail (CTT) from its large subunit, thus allowing proper insertion of nickel in the unique NiFe(CN)2CO cofactor present in these enzymes. The interaction of this tail with a dedicated endoprotease is dependent on the presence of the nickel atom which, at the same time, will be a key component of the enzyme active center. The presence of the CTT in the immature enzyme is essential for the adequate assembly of the enzyme.

In this work, a collaborative research involving two groups at the CBGP has carried out in silico analyses and Molecular Dynamics simulations to show that CTT in hydogenases are Intrinsically Disordered Regions (IDR) that likely provide the required flexibility to the protein for the final steps of proteolytic maturation. The model also shows that the presence of the CTT strongly modifies the environment of some key residues around the active site, as shown by the modification of the predicted pKa of specific residues in processed vs. unprocessed hydrogenase LSU. Furthermore, in silico and mutagenesis analysis have allowed the identification of a pair of conserved glutamate residues, one of which located at the CTT, that contribute to nickel incorporation into the enzyme, likely by providing an initial binding site for this metal atom. Structural details on the interaction of hydrogenase with the endoprotease responsible for the removal of CTT have been modelled.

These results obtained allow to get further insight on the biosynthesis of the hydrogenase active site, a key process for biotechnological applications focused on the use of hydrogen as a sustainable energy vector.