Glycerol-assisted Restorative Adjustment of Flavoenzyme Conformation Perturbed by Site-directed Mutagenesis

Abstract

The replacement of histidine 307 with leucine in pig kidney D-amino acid oxidase perturbs its active site conformation accompanied by dramatic losses in proteinflavin interactions and enzymatic activity. However, the negative effect of this mutation on the holoenzyme structure is essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flavin affinity. Further analysis revealed that glycerol assists in the rearrangement of the protein toward its holoenzyme-like conformation together with reduction in the solventaccessible protein hydrophobic area as demonstrated by limited proteolysis and use of affinity and hydrophobic probes. A substantial decrease in the protein-flavin interactions was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest that the perturbation of the D-amino acid oxidase active site is due to the nonpolar nature of the mutation whose negative impact on the holoenzyme structure can be overcome by glycerol-induced strengthening of protein internal hydrophobic interactions.The versatile properties of the flavin prosthetic group located in the active site of flavoenzymes have been successfully used over the years to study enzymatic redox mechanisms, including flavin-mediated activation of molecular oxygen (1, 2). In addition, the spectral redox properties and the reactivity of the flavin can be greatly manipulated by its modification with various chemical groups, a feature that has been particularly useful for structure functional analysis of flavoproteins (3-8). Flavoproteins also can be suitable models to study certain basic problems, such as protein stability, folding, and the effect of site-directed mutagenesis on protein conformation, since even the fine structural rearrangements within the protein active site can affect the flavin microenvironment and therefore be detected by perturbation of the binding mode and the spectral properties of the flavin. Recently, we employed this concept to demonstrate that glycerol, acting in vitro as a chemical chaperone, can assist in the proper refolding of the flavoprotein L-amino acid oxidase from its compact equilibrium intermediates and suggested the hydrophobic effect as a dominating force in this process (9). These findings prompted us to further investigate whether glycerol, in a similar manner, can induce proper adjustments of protein conformation perturbed by sitedirected mutagenesis, using a His-307 3 Leu (H307L) recombinant mutant of pig kidney D-amino acid oxidase (DAAO) 1 (10) as a model system. His-307 is one of two residues (the other is Tyr-228) undergoing affinity labeling with the D-propargylglycine-suicide substrate of pig kidney DAAO (11-13). Furthermore, the replacement of His-307 with Leu greatly reduces the protein affinity for FAD so that DAAO can be isolated only as an inactive apoprotein (14, 15). Although this is an indication that the presence of His-307 is important for protein...

Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

“…1), indicating that glycerol stabilizes the native CS structure. Glycerol-induced stabilization of intermediates has also been observed in Lamino-acid oxidase, in which glycerol is known to favor the hydrophobic network of partially folded structures and in turn stabilize the intermediates and favor the more compact state (71,72). Therefore, this suggests that glycerol may help form certain folding intermediates that are stabilized by hydrophobic interactions and help prevent intermolecular protein association.…”

Unmasking the Active Helicase Conformation of Nonstructural Protein 3 from Hepatitis C Virus

“…Glycerol stabilizes native protein structure by several mechanisms that are not mutually exclusive: (i) stabilization of the hydrophobic core, (ii) exclusion of glycerol from hydrophobic portions of a protein with preferential interactions with hydrophilic regions, and (iii) strengthening of hydrogen bonding networks (protein secondary structure) by preventing bulk water from acting as a hydrogen bond competitor (10,38,47). Increasing the glycerol concentration from 1% to 15% in our experimental conditions would be expected to perturb functional complex formation on RNA duplexes for WT NS3 by creating a larger kinetic barrier between the compact and extended conformations of the protein.…”

Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach

“…In green is reported the configuration corresponding the closed conformation (the complex of pkDAAO with benzoate, 1kif, and of RgDAAO with lactate, 1 cok), and in blue the one most closely resembling the open conformation (the complex of pkDAAO with iminotryptophan, 1ddo, and of RgDAAO with anthranilate, 1cOi). holoenzyme structure was essentially eliminated in the presence of glycerol [51]. The structural importance of His307 is also indicated by the fact that it is conserved in all DAAO sequences (see Fig.…”

“…In fact, the H307L pkDAAO mutant is purified as apoprotein [49][50][51]: its K d for FAD is 28-fold higher with respect to same value determined for the wild-type pkDAAO, while the activity is mostly retained. Although His307 is far from the cofactor (at ~11 Å), this residue is crucial for correct folding of the cofactor binding domain.…”