An enzymatic molten globule: efficient coupling of folding and catalysis

Abstract

A highly active, monomeric chorismate mutase, obtained by topological redesign of a dimeric helical bundle enzyme from Methanococcus jannaschii, was investigated by NMR and various other biochemical techniques, including H/D exchange. Although structural disorder is generally considered to be incompatible with efficient catalysis, the monomer, unlike its natural counterpart, unexpectedly possesses all of the characteristics of a molten globule. Global conformational ordering, observed upon binding of a transition state analog, indicates that folding can be coupled to catalysis with minimal energetic penalty. These results support the suggestion that many modern enzymes might have evolved from molten globule precursors. Insofar as their structural plasticity confers relaxed substrate specificity and/or catalytic promiscuity, molten globules may also be attractive starting points for the evolution of new catalysts in the laboratory.

Fig. 1.

Topological redesign of the enzyme MjCM. (A) The thermostable MjCM homodimer (Left) was converted into a monomer (mMjCM, Right) by inserting a flexible hinge loop (red) into the long H1 helix (9). The models are based on the x-ray structure (20) of a related E. coli chorismate mutase domain complexed with a transition-state analog, 1 (14), which is shown in the models in ball-and-stick representation. (B) Both enzymes efficiently catalyze the rearrangement of chorismate to prephenate.

Fig. 2.

NMR spectra of ¹⁵N-mMjCM and ¹⁵N-MjCM in the absence and presence of 1.(A) The [¹⁵N, ¹H]-TROSY spectrum of 40 μM mMjCM. Peak dispersion did not change appreciably when the protein concentration was varied in the range of 10 μM to 0.6 mM, ruling out oligomerization-induced line-broadening effects. (B) The [¹⁵N, ¹H]-TROSY spectrum of the ligand-bound mMjCM (sample from A, supplemented with 1.2 mM 1). The red numbers next to the peaks indicate the corresponding residues in the protein. The secondary structure of the monomer, assessed based on the chemical shifts of N, C^α, and C^β spins (P. Anikeeva and K.P., unpublished results), is in agreement with the proposed structural model (Fig. 1A). Residues 105–109 comprise part of the C-terminal His tag, which is unstructured and gives rise to degenerate resonances. (C) The [¹⁵N, ¹H]-TROSY spectrum of 0.6 mM MjCM. (D) The [¹⁵N, ¹H]-TROSY spectrum of the ligand-bound MjCM (sample from C, supplemented with 2 mM 1).

Fig. 3.

Characterization of mMjCM in the absence (magenta circles) and in the presence (cyan squares) of 1, and of MjCM in the absence (black diamonds) and in the presence (yellow triangles) of ligand. (A) Thermal denaturation curves for mMjCM with and without 1. The published (12) denaturation curve for MjCM is provided for comparison (scale on the right). (B) Near-UV CD spectra of the free and ligand-bound forms of mMjCM and MjCM. The well resolved peaks (280.5 and 287.5 nm) for the mMjCM·1 complex indicate that the two tryptophan residues located in the engineered loop are in a more asymmetric environment compared with the free protein (31). In the near-UV CD spectrum of MjCM (which contains no tryptophans) these peaks are not observed. (C) Fluorescence emission spectra of the free and ligand-bound proteins in the presence of ANS and of ANS alone (green triangles).

Fig. 4.

H/D exchange of mMjCM (magenta circles), mMjCM·1 (cyan squares), MjCM (black diamonds), and MjCM·1 (yellow triangles). The substrate chorismate protects mMjCM from exchange to a similar extent as 1 (data not shown).