Directed evolution of a (βα) ₈ -barrel enzyme to catalyze related reactions in two different metabolic pathways

Abstract

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...

Mimicking natural evolution in vitro : An N -acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

“…By rational design we could enable NAL from E. coli to replace an enzyme from another pathway in vivo while still maintaining much of its original activity. A similar scenario has been described for two structurally similar (␤͞␣) 8 -enzymes from the biosynthetic pathways of histidine and tryptophan, where random mutagenesis on one of these two homologs yielded a mutant exhibiting both activities (33). Our findings also offer a structural rationalization for the observed activities.…”

Divergent Evolution of (??)8-Barrel Enzymes

“…Here we summarize recent sequence and structure analyses, as well as protein engineering studies, which provided new insights into the evolution of (βα) 8 -barrels. The results suggest that the members of several important superfamilies and probably a large fraction of all known (βα) 8 -barrels have a common evolutionary origin (Altamirano et al, 2000;Babbitt and Gerlt, 2000;Copley and Bork, 2000;Jürgens et al, 2000). Moreover, evidence for the evolution of the (βα) 8 -barrel fold from ancestral 'halfbarrel' precursors will be presented (Thoma et al, 1998;Lang et al, 2000;Höcker et al, 2001).…”

“…Using random mutagenesis and selection in a trpF-deficiency strain, HisA variants were generated that catalysed the TrpF reaction. Moreover, one of these variants retained significant HisA activity (Jürgens et al, 2000). A closer analysis revealed that a single amino acid exchange in the active site region was sufficient to interconvert the substrate specificity from HisA to TrpF, although the enzymes share a sequence identity of only about 10%.…”

Using multi-objective computational design to extend protein promiscuity

“…Proteins with several activities (multipurpose enzymes) may have a wide range of biotechnological applications related (but not limited) to industrial organic synthesis and metabolic engineering [12][13][14][15][16][17]. However, natural protein promiscuity (as described in the two preceding paragraphs) may perhaps be of limited use in the development of these multipurpose catalysts.…”