It is well-known that the large diversity of protein functions and structures derives from the broad spectrum of physicochemical properties of the 20 canonical amino acids that make up modern proteins. According to the generally accepted hypothesis [1], evolution of protein structures and functions was continuously associated with enrichment of the genetic code, with aromatic amino acids being considered the latest addition to the genetic code to increase structural stability of proteins and enhance clarification of its catalytic functions [2].
The main objective of this study was to test whether enzymatic catalysis can spare the structural stability provided by aromatics by determining the effect of amino acid alphabet reduction on structure and function of dephosphoCoA kinase (DPCK). Two mutant variants of a putative DPCK from Aquifex aeolicus (PDB code 2IF2) without aromatic amino acids were designed, substitution by Leu residues (DPCK-LH) and by non-aromatic residues based on the best predicted preservation of thermodynamic stability (DPCK-M).
Structural characterization of DPCK variants (using CD and 1D-NMR) suggest that there is less tertiary structure and partial loss of secondary structure content upon loss of the aromatic residues. Intriguingly, DPCK-LH seems to be structurally more similar to DPCK-WT than the DPCK-M variant. The results of structural studies reflect on catalytic activities of protein variants with the gradual decrease of protein folding echoing in the steady loss of substrate specificity in a row DPCK-WT – DPCK-LH – DPCK-M. Whereas DPCK-WT shows phosphotransferase activity, DPCK-LH and DPCK-M rather demonstrate ATPase activity and 150 and 500 times lower catalytic efficiency in ATP conversion, respectively, mainly as a result of a decreased turnover number. The HPLC-MS analysis detected significant CoA formation only in the reaction catalysed by DPCK-WT but 10x and 100x less formation was detected also in the reactions catalysed by DPCK-LH and DPCK-M, respectively.
The obtained data provide evidence to the hypothesis that proteins in the early stages of life could support at least some enzymatic activities, probably with lower efficiencies as a result of lack of firm hydrophobic core.
We would like to thank Dr. Radko Souček for help with the amino acid analysis. This work was supported by the Czech Science Foundation (GAČR) grant number 17-10438Y, project SVV260427/2018 (VT) and by the project “BIOCEV” CZ.1.05/1.1.00/02.0109. We would also like to acknowledge the facility and support of the CMS-Biocev ("Biophysical techniques”) supported by MEYS ČR (LM2018127).