Evaluation of the Conformational Stability of Recombinant Desulfurizing Enzymes from a Newly Isolated Rhodococcus sp.

Metabolic pathways of aerobic bacteria able to assimilate sulfur can provide biocatalysts for biodesulfurization of petroleum and of other sulfur-containing pollutants. Of major interest is the so-called "4S pathway," in that C-S bonds are specifically cleaved leaving the carbon skeleton of substrates intact. This pathway is carried out by four enzymes, named Dsz A, B, C, and D. In view of a possible application of recombinant Dsz enzymes in biodesulfurization treatments, we have investigated the structural features of enzymes cloned from a Rhodococcus strain isolated from polluted environmental samples and their resistance to temperature (20-95 °C) and to organic solvents (5, 10, and 20 % v/v methanol, acetonitrile, hexane, and toluene). Changes in protein structures were assessed by circular dichroism and intrinsic fluorescence spectroscopy. We found that all Dsz proteins are unfolded by temperatures in the range 45-60 °C and by all solvents tested, with the most dramatic effect being produced by toluene. These results suggest that stabilization of the biocatalysts by protein engineering will be necessary for developing biodesulfurization technologies based on Dsz enzymes.

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods.

Protein structure and dynamics are crucial for protein function. Thus, the study of conformational properties can be very informative for characterizing new proteins and to rationalize how residue substitutions at specific protein sites affect its dynamics, activity and thermal stability. Here, we investigate the structure and dynamics of the recently isolated cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila (SpAAP) by the integration of simulations, circular dichroism, mass spectrometry and other experimental data. Our study notes traits of cold-adaptation, such as lysine-to-arginine substitutions and a lack of disulphide bridges. Cold-adapted enzymes are generally characterized by a higher number of glycine residues with respect to their warm-adapted counterparts. Conversely, the SpAAP glycine content is lower than that in the warm-adapted variants. Nevertheless, glycine residues are strategically located in proximity to the functional sites in SpAAP, such as the active site and the linker between the two domains.. In particular, G457 reduces the steric hindrance around the nucleophile elbow. Our results suggest a local weakening of the intramolecular interactions in the cold-adapted enzyme. This study offers a basis for the experimental mutagenesis of SpAAP and related enzymes. The approaches employed in this study may also provide a more general framework to characterize new protein structures in the absence of X-ray or NMR data.

Reciprocal influence of protein domains in the cold-adapted acyl aminoacyl peptidase from Sporosarcina psychrophila.

Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/ß-hydrolase domain and by an N-terminal ß-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the ß-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated ß-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.