Characterization of the stabilizing effect of point mutations of pyruvate oxidase from lactobacillus plantarum: Protection of the native state by modulating coenzyme binding and subunit interaction

Abstract

Point mutations in the gene of pyruvate oxidase from Lactobacillusplantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions,or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased.The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 "C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.

Hydrophobic Core Substitutions in Calbindin D9k: Effects on Stability and Structure

“…Notable is also that the intercepts are negative, i.e., mutations changing one residue to another of the same volume, surface area, or hydrophobicity on average lead to destabilization. This result is in line with observations on random point mutations that indicate that in general only one out of 10 3 -10 4 mutations will lead to increased protein stability (62)(63)(64)(65)(66). It would appear that most point mutations in the hydrophobic core of a protein will cause a perturbation that can be related to different factors such as ∆A, ∆V, or ∆∆G trans of the mutated residues.…”

Stability and reconstitution of pyruvate oxidase from lactobacillus plantarum: Dissection of the stabilizing effects of coenzyme binding and subunit interaction

“…In an accompanying paper we describe pyruvate oxidase mutants that are stabilized by single or multiple point mutations (Risse et al, 1992). For the analysis of these proteins, one has to keep in mind that stabilizing point mutations may be effective at the level of the tertiary or the quaternary structure, or by modulating cofactor binding.…”

The Concept of Solvent Compatibility and Its Impact on Protein Stability and Activity Enhancement in Nonaqueous Solvents

“…Because little is known regarding the mechanism(s) by which polar solvents reduce the activity of soluble enzymes, random mutagenesis approaches have often been used to improve the catalytic performance of proteins. 26,27 Random mutagenesis and screening has been used to enhance or alter various enzyme features, including enhanced thermal stability, [28][29][30][31][32] alkaline stability, 33 substrate specificity, 34 higher activity in organic solvents, 35 and recovering the catalytic activity of an enzyme damaged by site-directed mutagenesis. 36 Although proteins in organic solvents have been extensively studied, 37 their amino acid sequences and even their three-dimensional structures do not provide us clear insights into how to confer desirable properties on these systems (e.g., enhanced activity or stability in a variety of solvent systems).…”