Computational analyses,molecular dynamics,and mutagenesis studies of unprocessed form of [NiFe] hydrogenase reveal the role of disorder for efficient enzyme maturation |
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Authors: | Marta Albareda Luis F Pacios Jose M Palacios |
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Institution: | Centro de Biotecnología y Genómica de Plantas (C.B.G.P.) UPM-INIA, Universidad Politécnica de Madrid, Campus de Montegancedo, 28223 Pozuelo de Alarcón, Spain;Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain |
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Abstract: | Biological production and oxidation of hydrogen is mediated by hydrogenases, key enzymes for these energy-relevant reactions. Synthesis of NiFe] hydrogenases involves a complex series of biochemical reactions to assemble protein subunits and metallic cofactors required for enzyme function. 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. In silico modelling and Molecular Dynamics (MD) analyses of processed vs. unprocessed forms of Rhizobium leguminosarum bv. viciae (Rlv) hydrogenase large subunit HupL showed that its CTT (residues 582-596) is an intrinsically disordered region (IDR) that likely provides the required flexibility to the protein for the final steps of proteolytic maturation. Prediction of pKa values of ionizable side chains in both forms of the enzyme's large subunit also revealed that the presence of the CTT strongly modify the protonation state of some key residues around the active site. Furthermore, MD simulations and mutant analyses revealed that two glutamate residues (E27 in the N-terminal region and E589 inside the CTT) likely contribute to the process of nickel incorporation into the enzyme. Computational analysis also revealed structural details on the interaction of Rlv hydrogenase LSU with the endoprotease HupD responsible for the removal of CTT. |
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Keywords: | IDR intrinsically disordered region LSU large subunit SSU small subunit CTT C-terminal tail from hydrogenase LSU MD molecular dynamics RMSD root mean square deviation RMSF root mean square fluctuation Metalloenzyme Nitrogen fixation Hydrogenase Molecular modelling C-terminal tail [NiFe] cofactor |
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