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

Abstract

In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific singlesubstrate enzymes (HisA and TrpF), sharing a similar ðβ∕αÞ 8 -barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme.amino acid biosynthesis | enzyme evolution | mycobacteria | active site substrate adaptability | protein structure symmetry

Structural and functional innovations in the real-time evolution of new (βα) 8 barrel enzymes

“…3B). A different arginine, Arg143, has an equivalent interaction with rCdRP in the active site of PriA (11). Another S. enterica mutant with TrpF activity, HisA(L169R) (8), places Arg169 in a similar position (Fig.…”

“…3C and SI Appendix, Table S2), as does TrpF itself (14). The appropriate positioning of an arginine appears to be a shared innovation for PRA binding and a catalytically competent active site (11,14) in the emergence of TrpF activity on the (βα) 8 barrel scaffold.…”

“…A HisA(W145A) mutant was unable to rescue the histidine auxotrophy of S. enterica ΔhisA, showing that the Trp145-ProFAR interaction is essential for ProFAR isomerase activity. Mutagenesis of the equivalent tryptophan in PriA also destroys HisA activity (11). The D10G mutation in loop 1, which reintroduced HisA activity into HisA(dup13-15), does not induce any changes in the crystal structure, but is predicted to allow more flexibility of loop 1, so that loop 5 can compete for the active site (Fig.…”

“…with catalytic efficiencies on the order of 10 2 -10 3 s −1 M −1 conferred maximal growth rates. In comparison, the Escherichia coli TrpF enzyme has k cat /K M = 6.8 × 10 6 s −1 M −1 (25) and the bifunctional M. tuberculosis PriA has k cat /K M = 1.7 × 10 5 s −1 M −1 for the TrpF reaction (11). In E. coli, TrpF is synthesized at a rate of ∼110 molecules per generation when tryptophan is present in the medium, rising by approximately eightfold when the trp operon is derepressed in minimal medium (26).…”

Enzyme evolution beyond gene duplication

“…11,13,14 At the center of this model stands a dual-substrate (βα) 8 -barrel phosphoribosyl isomerase (priA gene), which is a close homolog (~50% ID) of the histidine biosynthetic hisA gene. 12, 15 The discovery of PriA stemmed from the realization that after whole genome sequencing of these organisms the trpF gene could not be found.…”