Two‐fold repeated (βα) ₄ half‐barrels may provide a molecular tool for dual substrate specificity

Abstract

Some bacterial genomes contain an incomplete set of genes encoding phosphoribosyl isomerases, raising the question of whether there exists broadened substrate specificity for the missing gene products. To investigate the underlying molecular principles of this hypothesis, we have determined the crystal structure of the bifunctional enzyme PriA from Streptomyces coelicolor at 1.8 Å resolution. It consists of a (ba) 8 -barrel fold that is assembled by two symmetric (ba) 4 half-barrels. The structure shows how its active site may catalyse the isomerization reactions of two different substrates, and we provide a plausible model of how the smaller of the two substrates could be bound in two different orientations. Our findings expand the half-barrel ancestor concept by demonstrating that symmetry-related halfbarrels could provide a smart solution to cope with dual substrate specificity. The data may help to unravel molecular rationales regarding how organisms with miniature genomes can keep central biological pathways functional. Keywords: phosphoribosyl isomerase; evolution of (ba) 8 -barrels; tryptophan and histidine biosynthesis EMBO reports (2005) 6, 134-139.

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

“…Our data, however, do not support previous hypotheses on either the potential use of the twofold repeated half-barrel symmetry or general loop flexibility considerations, which present features largely shared by the bisubstrate PriA enzyme and the single-substrate HisA enzyme (12,14). These hypotheses were raised based on available apo PriA structures only, without experiment-based knowledge of the type of conformational changes associated with substrate binding.…”

“…The observations point to a close evolutionary relationship between PriA and HisA. However, in contrast to available apo structures of PriA from M. tuberculosis (this contribution) and S. coelicolor (12,13), in which Asp175 is either remote from the active site or invisible, requiring recruitment into the active site upon substrate binding, in the available apo structure of HisA from T. maritima (9), the equivalent residue (Asp169) is oriented into the active site (Fig. S6A).…”

“…S1 A and B and Table S1). Comparison of this structure with those of the same enzyme from S. coelicolor in the presence of sulfate (12,13) reveals no significant changes of the overall fold and active site loop structure, indicating that the conformational changes observed in the two PriA-ligand complexes are caused by the presence of the reaction ligands.…”

“…A hisA-like gene, referred to as priA, was shown to encode an enzyme with bisubstrate specificity in Streptomyces coelicolor, rendering a bifunctional his/trp biosynthesis isomerase (11). Although some active site residues from the C-terminal ðβ∕αÞ 8 -barrel face in PriA have been shown to be involved in PriA catalysis (12)(13)(14), it was not known which molecular mechanism renders PriA a bisubstrate enzyme, in the absence of structural insight into the positioning of any of the two substrates within the active site. Here, we have investigated the PriA enzyme from Mycobacterium tuberculosis to resolve this question.…”

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…Although the sequence identity between TrpF and HisA is only Ϸ15%, the successful design of detectable PRA isomerisation activity by exchanging only a few HisA residues suggests that the 2 (␤␣) 8 -barrel enzymes have evolved from a common ancestor by gene duplication and diversification (9). In further support of this hypothesis, a naturally bifunctional isomerase (PriA) has been identified in several microorganisms that catalyses both the TrpF and the HisA reaction with high efficiency (16,17).…”