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...

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The aim of this study was to increase the stability of the thermolabile (ba) 8 -barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges. For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines. The variant linking residues 3 and 189 fastens the N-terminus to the (ba) 8 -barrel. The rate of thermal inactivation at 50°C of this variant with a closed disulfide bridge is 65-fold slower than that of the reference dithiol form, but only 13-fold slower than that of the parental protein. The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified. To confirm these data, we have solved the X-ray structure to 2.1-Å resolution. The second variant was designed to crosslink the terminal modules ba1 and ba8. However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein.Keywords: indoleglycerol phosphate synthase; (b/a) 8 -barrel proteins; stabilizing disulfide bonds; protein engineering.Indoleglycerol phosphate synthase (IGPS) is a (ba) 8 -barrel protein with an N-terminal extension of 48 residues. In Escherichia coli, IGPS (eIGPS) is the N-terminal domain of a monomeric, bifunctional enzyme, where the C-terminal domain is phosphoribosyl anthranilate isomerase (ePRAI), folded into another (ba) 8 -barrel [1]. The catalytic efficiencies of the engineered separated domains are virtually identical to those in the bifunctional enzyme [2]. eIGPS is, however, more labile than ePRAI. The catalytic activity of eIGPS decays at 55°C with a half-life of 0.5 min [3]. In contrast, ePRAI activity decays at 60°C with a half-life of 100 min (R. Sterner, Institut fu¨r Biochemie, Universita¨t zu Kö ln, Germany, personal communication). The eIGPS domain, in turn, is also more labile than eIGPS in the native bifunctional protein [4,5,6].In contrast to eIGPS [1], the IGP synthases from the hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and Thermotoga maritima (tIGPS [3]), are thermostable, monofunctional monomers. The comparison of the three high resolution crystal structures suggests that an increased number of salt bridges over that in eIGPS decreases the rates of irreversible thermal inactivation of both sIGPS and tIGPS. In support of this proposal, mutational disruption of salt bridge that crosslinks its terminal ba1 and ba8 modules, significantly destabilized the variants by comparison to the parental enzyme [3], in support of analogous findings reported previously [8].The aim of this work was to stabilize the labile eIGPS domain by introducing new disulfide bonds rather than new salt bridges. Disulfide bonds can stabilize p...