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 single-substrate enzymes (HisA and TrpF), sharing a similar (β/α)(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.

Fig. 1.

Ligand-induced active site changes of PriA. (A) Active site loop structure of the PriA complexes with sulfate (Left), rCdRP (Center), and PrFAR (Right). The eight active site loops 1–8 are colored in yellow (1,5), green (2,6), cyan (3,7), and magenta (4,8), emphasizing the twofold repeated (β/α)₄ half-barrel elements (9); those loop segments that are disordered are indicated by dashed lines. The remaining structures are shown in surface presentation (β-strands, light gray; α-helices, dark gray). Each ligand is shown in stick presentation. (B) Ligand-specific Asp175 recruitment: by Arg143, in the presence of rCdRP (trp biosynthesis, Left); and by Arg19, in the presence of PrFAR (his biosynthesis, Right). Loop 5 changes from a β-hairpin conformation (rCdRP) to a knot-like conformation (PrFAR), allowing Arg143 and Trp145 to switch positions. Hydrogen bonds are shown with dashed lines. The areas shown in B approximately correspond to the red boxes in A.

Fig. 2.

Active site structures of the PriA-rCdRP and PriA-PrFAR complexes. Each ligand (rCdRP, A; PrFAR, B) is shown in 2Fo-Fc electron density at a 1,5 σ contour level. Hydrogen bonds between each ligand and PriA residues, in part mediated by ordered solvent molecules (red spheres), are represented with dashed lines. Highlighted residues and ligands are shown in stick presentation, using atom-specific colors (oxygen, red; nitrogen, blue; sulfur, yellow; phosphorus, magenta). The carbon atoms of PriA residues are colored according to the scheme used in Fig. 1; carbon atoms of ligands are in gray. Residues involved in side chain-specific interactions with ligands are labeled. The labels of those residues that have been identified in catalysis or ligand-induced active site recruitment are colored red. The remaining part of each PriA structure is shown in ribbon representation.

Fig. 3.

Biochemical analysis of PriA ProFAR/PRA isomerization. Relative catalytic efficiencies of several PriA variants (wild-type PriA = 100%), in which active site residues were mutated into alanines: ProFAR isomerization, black bars; PRA isomerization, gray bars (A). The complete data are listed in Table S2. The effects of the PriA mutations on ProFAR isomerization (B) and PRA isomerization (C) are graphically mapped onto the respective PriA-ligand complexes. The color codes of the mutated residues, shown in ball-and-stick presentation, are red, < 1% wt activity; orange, < 5% wt activity; green, < 50% wt activity; blue, > 50% wt activity. The orientation of PriA is as in Fig. 1.