Indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase: Comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains

Abstract

Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.

A Study in Molecular Contingency: Glutamine Phosphoribosylpyrophosphate Amidotransferase is a Promiscuous and Evolvable Phosphoribosylanthranilate Isomerase

“…In E. coli, PRAI forms the C-terminal domain of a bifunctional IGPS:PRAI enzyme; however, the IGPS and PRAI domains can be separated genetically and expressed as stable, monomeric proteins, each with essentially wild-type catalytic activity. 21 PRA isomerization is a good model reaction for several reasons. First, the frequent occurrence of (βα) 8 -barrel proteins within the protein database suggests that this scaffold may be particularly evolvable.…”

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

“…The source of the etrpC gene for the monofunctional, monomeric elGPS domain ) was the plasmid pMc2.C [6] …”

“…The yields of purified proteins (mg protein per g of wet cell paste) were: sIGPS, 1.5; sA(2-9), 0.31. Wild-type elGPS-PRAI was purified as described [6]. Because the expression of the genes of elGPS (IGPS [l-259], [6]), eL5V and eA(2-9)-PRAI employed the efficient expression vector pDS56/RBSII/SpM [16], these proteins were recovered from the insoluble fraction of cell homogenates, and were already about 90% pure.…”

“…These are phosphoribosyl anthranilate isomerase (PRAI, [5,6]), indoleglycerol phosphate synthase (IGPS, [5,7]) and the a sub unit of tryptophan synthase [8]. Each of the three enzymes is in principle a stable and active monomer [6][7][8][9].…”

“…Here we used protein engineering and enzyme kinetics to probe the role of the extra helix ao of the recombinant monofunctional IGPS domain from Escherichia coli (elGPS [6]) by replacing the invariant leucine residue in the first turn by valine as well as by deleting the N-terminal portion of the helix a 0 . Because the deletion destabilizes elGPS, the same deletions were constructed in both the bifunctional enzyme elGPS-PRAI and in the thermostable, monofunctional IGPS from Sulfolobus solfataricus (sIGPS [7,9]).…”