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

Abstract

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-l-deoxyribulose 5 ' -phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state ktnetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.

Imidazole Glycerol Phosphate Synthase fromThermotoga maritima

“…where p is the probability that, for m randomized codons appearing with the relative frequency f i , practically all possible amino acid combinations are present in a library containing n independent clones (23).…”

The Crystal Structure of Indoleglycerol-phosphate Synthase from Thermotoga maritima

“…Five of these identical residues are invariant or highly conserved among 25 sequences. Two of the invariant residues are catalytically essential in eIGPS (Lys 114 and Glu 163 ), and one (Glu 53 ) is catalytically important (38). The remaining five positions are occupied by similar residues (i.e.…”

“…5, a and b). One is between the catalytic residues Glu 47 and Lys 108 in tIGPS (Glu 51 and Lys 110 in sIGPS), which are buried in the active site, and where the mutual orientation of their general acid/base groups is likely to be functionally important (38). This salt bridge is also conserved in eIGPS (Glu 53 -Lys 114 ).…”

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase

“…Based on the available crystal structures and by modeling the intermediate in the enzyme active site (In), Kirschner and coworkers (5) provided useful insights into the catalytic mechanism of IGPS. The general acid and the general base for the reaction have been proposed to be Lys-110-NH 3 ϩ and Glu-159-CO 2 Ϫ , respectively (6). However, in the x-ray crystal structures of IGPS bound to the substrate (CdRP) and substrate analogue, both ligands are bound in an extended, unproductive conformation, such that the two reactive carbon centers C1 and C2Ј are separated by a distance that is too large to initiate bond formation.…”