Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes

Abstract

The role of the extra helix do in the N-terminal extension of the eight-fold ßa barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation. Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither k cat nor K M , but deletion of 8 N-terminal residues decreased solubility strongly. The similarly truncated variant from the hyperthermophile Sulfolohus solfataricus was soluble, and had the same k cat value as the wild-type protein but a 220-fold greater K M value. These results suggest that the Nterminal portion of helix Oo provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state. Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold ßa barrels, as required for their divergent evolution.

Directed evolution of new catalytic activity using the α/β-barrel scaffold

“…This mutant was unstable, had a tendency to aggregate 31 and was catalytically inactive with respect to both IGPS and PRAI. This mutant was unstable, had a tendency to aggregate 31 and was catalytically inactive with respect to both IGPS and PRAI.…”

Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

“…The active site lies between the phosphate group binding site, which is near the C-termini of p7 and ps, and the hydrophobic pocket formed by the following invariant hydrophobic side-chains: L5 on helix a. (Stehlin et al, 1997), the invariant segment S58P59S60 located on loop p I a I (15 residues long), F116 on p3a3 (5 residues), and L188 on &,a6, (10 residues). The bound phosphate ion and the sidechains of K55, E53, and K114 form a cluster of salt bridges on one wall of the catalytic center.…”

“…Both functional domains display the same (pa)x-barrel fold as the archetypal triose phosphate isomerase. Because both eIGPS and ePRAI possess structurally identical Pa(7-8) subdomains for binding the phosphate moieties of the respective substrates and products, it is possible that they have evolved from a common ancestor (Farber & Petsko, 1990;Wilmanns et al, 1991;Stehlin et al, 1997). Moreover, the substrate of the IGPS reaction (CdRP, Fig.…”

Stabilization of a (βα)8‐barrel protein by an engineered disulfide bridge

“…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 contrast, ePRAI activity decays at 60 °C with a half‐life of 100 min (R. Sterner, Institut für Biochemie, Universität zu Köln, Germany, personal communication). The eIGPS domain, in turn, is also more labile than eIGPS in the native bifunctional protein [4,5,6].…”