Enzyme catalysis prior to aromatic residues: reverse engineering of a dephosphoCoA kinase

Abstract

It is well-known that the large diversity of protein functions and structures is derived from the broad spectrum of physicochemical properties of the 20 canonical amino acids. According to the generally accepted hypothesis, protein evolution was continuously associated with enrichment of this alphabet, increasing stability, specificity and spectrum of catalytic functions. Aromatic amino acids are considered the latest addition to genetic code. The main objective of this study was to test whether enzymatic catalysis can spare the aromatic amino acids (aromatics) by determining the effect of amino acid alphabet reduction on structure and function of dephospho-CoA kinase (DPCK). We designed two mutant variants of a putative DPCK from Aquifex aeolicus by substituting (i) Tyr, Phe and Trp or (ii) all aromatics (including His), i.e. [~]10% of the total sequence. Their structural characterization indicates that removal of aromatic amino acids may support rich secondary structure content although inevitably impairs a firm globular arrangement. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate is however heavily uncoupled and only one of the variants is still able to perform the reaction. Here we provide support to the hypothesis that proteins in the early stages of life could support at least some enzymatic activities, despite lower efficiencies resulting from the lack of a firm hydrophobic core. Based on the presented data we hypothesize that further protein scaffolding role may be provided by ligands upon binding. SignificanceAll extant proteins rely on the standard coded amino acid alphabet. However, early proteins lacked some of these amino acids that were incorporated into the genetic code only after the evolution of their respective metabolic pathways, aromatic amino acids being among the last additions. This is intriguing because of their crucial role in hydrophobic core packing, indispensable for enzyme catalysis. We designed two aromatics-less variants of a highly conserved enzyme from the CoA synthesis pathway, capable of enzyme catalysis and showing significant ordering upon substrate binding. To our knowledge, this is the first example of enzyme catalysis in complete absence of aromatic amino acids and presents a possible mechanism of how aromatics-less enzymes could potentially support an early biosphere.