Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N -[(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (␤␣) 8-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (␤␣) 8-barrel enzymes are very suitable for the design of new catalytic activities.E nzymes of contemporary metabolic pathways are generally specific and efficient biocatalysts. They can be categorized into a limited number of families, the members of which share similar reaction mechanisms, folds, or both (1). This leads to the idea that the members of a given enzyme family are evolutionarily related. In principle, two different evolutionary scenarios can be envisioned. New catalytic functions of enzymes could have evolved by changing the chemistry of catalysis, while retaining the binding capacity for a common ligand. This idea of retrograde evolution (2) was recently supported by the successful interconversion of the catalytic activity of two enzymes from tryptophan biosynthesis, which catalyze successive reactions in the pathway and therefore bind the same ligand (3). Alternatively, new catalytic functions could have evolved by retaining the chemistry of catalysis, while changing the substrate specificity (1). Along these lines, the patchwork model of enzyme evolution (4) postulates that ancestral enzymes were relatively unspecific and therefore were capable of catalyzing chemically similar reactions in different metabolic pathways. Genes encoding these enzymes would have duplicated in the course of evolution and would have subsequently specialized by diversification. NЈ-[(5Ј-Phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24) constitute a pair of similar enzymes, which are involved in the biosynthesis of the amino acids histidine and tryptophan, respectively (5, 6). Both HisA and TrpF catalyze an Amadori rearrangement, which is the irreversible isomerization of an aminoaldose to an aminoketose (Fig. 1). Also, despite the lack of detectable amino acid sequence similarity, HisA and TrpF belong to the same structural family of (␤␣) 8 -barrels (7, 8), which is the most frequent fold among single-dom...

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNAPro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNAPro is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In the present study, experimental and computational approaches were used to test the hypothesis that INS deletion alters the internal protein dynamics leading to reduce catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199–206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled-dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Taken together, the present study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.