The folding and assembly of the dodecameric type II dehydroquinases

Abstract

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)‐2‐Benzyl‐3‐dehydroquinic Acids

“…Simulations were carried out using the enzyme geometries found in the crystal structures of DHQ2‐Mt and DHQ2‐Hp in complex with p ‐methoxybenzyl derivative 4 a (PDB: 2XB8 and 2XB9, respectively). Taking into account that unfolding and refolding studies of DHQ2 have shown that the trimer38 is the minimal biologically active unit of the enzyme, and on the basis of preliminary simulations that showed the monomer to be unstable under our simulation conditions, the trimer was used for these studies. Hydrogen atoms were added to the protein using web‐based H++ server,40 which assigned protonation states to all titratable residues at the chosen pH of 7.0.…”

A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

“…Distinct from type I enzymes, type II DHQDs are found exclusively within bacteria. These ~17 kDa enzymes assemble into homo-dodecamers and catalyze a reaction that proceeds via a non-covalent enolate intermediate to result in a trans-elimination (12–14). …”

Nitrilase ofRhodococcus rhodochrousJ1

“…For example, a pigeon liver malic enzyme, which is either composed of a monomer or a dimer, was associated with the fully active tetramer by basic pH and high temperatures or ionic strength, but was dissociated by increased ammonium sulfate concentrations of 0.2 m [32]. Further examples are a RecA protein, which self‐assembled with increased ion and protein concentrations [33], and the dissociated form of a dehydrogenase, that aggregated to the active dodecamer by high enzyme concentrations [34].…”