46907-GB4
Development of Peptide Based Biocatalysts for the Desulfurization of Dibenzothiophene
The combustion of sulfur generates sulfur oxyacids that contribute to deforestation and cardiopulmonary disease. The current technology for reducing sulfur content in fuels, hydrodesulfurization, cannot eliminate recalcitrant aromatic sulfur compounds. Discovery of bacteria that metabolize aromatic sulfur heterocycles has led to significant interest in biocatalytic desulfurization. However, meeting the aqueous environment needed by the organism, while effectively removing sulfur from fuel stocks has not been achieved.
In the biological metabolism of DBT usas DszB, a desulfurase, and is thought to involve a number of flavodoxins, or related enzymes. The crystal structure of DszB, from Rhodococcus erythropolis, was recently solved, revealing that binding occurs from contact with six primary residues, three of which display an edge-face aromatic interaction. Flavodoxin binding to flavin commonly involves aromatic residues as well. Favorable electrostatic interactions arise from the proximity of the electron rich centroid of the ring and the electron poor hydrogens. The ubiquitous presence of aromatic interactions in enzymatic action on DBT has biased design of a molecular receptor.
The peptide biocatalyst includes a number of essential features: a regular structure, modifiable sidechains and aromatic residues to interaction with DBT. Progress towards peptide receptor exploration has resulted in four possible sequence designs, cyclic and acyclic versions of diagonal and lateral sidechains. Initially, in order to guarantee structural rigidity all peptides were macrocyclized with a disulfide bond from terminal cystine residues. However, many peptides demonstrated sufficient stability to consider eliminating the disulfide bond to allow for greater positional freedom. The shift from random coil of the Ha resonances are typically between 0.05 and 0.7 ppm with more than a 0.1 ppm shift considered an indication of structure. Disulfide bonds are still used in the event that a peptide is not sufficiently folded. In this manner greater conformational flexibility is available to maximize interactions with a substrate.
The positioning of the aromatic sidechains has been varied to determine the impact on binding site. Initially the aromatic residues were positioned diagonally (i, j-2) within the peptide. A number of peptides were synthesized and characterized utilizing this structural motif (RFVKVNGOFIKQ, RWVTVNGOWITQ, RWVKVNGOWIKQ, and RWVKVNGOWIFQ). All of these peptides were well folded (greater than 60%) and have been characterized. A number of technical problems have been found from early investigations into the binding potential of these peptides. The excitation band for Trp is altered within the diagonal geometry such that it overlaps with the DBT absorbance band. As such binding data cannot be obtained from fluorescence spectroscpoy. Anaylsis with NMR based binding studies in much slower and ongoing. The phenylalanine containing peptides are expected to bind the weakest, but could be amenable to fluorescence analysis and will be investigated. The aromatic rings are not sufficiently close to interaction with one another, but can bind to an intercalated substrate in a sandwich motif. While this offers an efficient method for packing it appears to confer only offset stacked geometries.
To diversify the accessible geometries for interacting with substrates a variation is being explored. Laterally disposed aromatic residues (i, j) are sufficiently close to interact with each other, which seems to preclude them from subsequent substrate interactions. However, our studies indicate that the lateral interaction occurs predominantly through one or two hydrogens via an edge-face geometry. The shift of specific aromatic resonances from random coil, when laterally positioned with a phenylalanine, are between 0 and 1.4 ppm. What is interesting from a design perspective is that 1-naphthylalnine and tryptophan have very similar trends with the 7, 8 and 4, 5 hydrogens, respectively, the most upfield shifted. This suggests that these two rings adopt similar geometries. A subtle shift in the point of attachment to 2-naphthylalanine gives a very different set of shifts, wher the 1, 3 positions are most shifted. For both 1- and 2-naphthylalanine the majority of the aromatic ring is not involved in a lateral aromatic interaction. A preliminary geometry suggests that the aromatic rings are edge-face with a cleft for edge-face type interactions with a substrate.
Future direction: Analysis of the binding of peptide to DBT and DBTO2 with the diagonal aromatic residues is ongoing. A computational structure will be generated for the lateral polycyclic aromatic peptides from NOE restraints. Binding data will then be generated with the lateral aromatic peptides. After this initial round of diagonal and lateral structural and binding analysis an investigation into the redox potential of DBT and perturbations from binding will begin.
