|Ph.D Student||Shainsky-Roitman Janna|
|Subject||Structure-Function Correlations of Mono-and Di-Oxygenases|
and Pro-Chiral Sulfides
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Ayelet Fishman|
|Full Thesis text|
The main goal of the present research was to investigate the correlation between the structure of pro-chiral sulfides and the activity/selectivity of two families of oxygenases, toluene monooxygenases (TMOs) and nitrobenzene dioxygenase (NBDO).
In the first part of the research four different methods were developed for detecting sulfoxides that were produced via whole cell biocatalysis, and two of them, were further exploited for high throughput screening. One method is based on the reduction of sulfoxides to sulfides with the coupled release and measurement of iodine. The second method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants towards (S)-methyl-p-tolyl sulfoxide.
The second part of the work included the investigation of two residues in toluene-4-monooxygenase (T4MO): TmoA D285 is located at the entrance of the tunnel leading to the active site, while residue I100 is positioned in the vicinity of the diiron center and is part of the active site pocket. It was discovered that mutations at position 285 enhanced the rate of catalysis but did not affect the specificity. In contrast, mutations at position I100 influenced the activity rate and enantioselectivity. Combining the beneficial effect of both substitutions resulted in an additive or even synergistic outcome on enzyme activity and selectivity.
Position TomA3 V106 in TOM was previously reported as influencing activity and selectivity of the enzyme. In this work, the range of the prochiral substrates catalyzed by TOM was broadened. It was shown that changing Val to another residue influenced activity and selectivity especially for large substrates. It is suggested that the physical shape and hydrophobic forces of the active site pocket were changed, and therefore the substrate was oriented differently resulting in higher activity and selectivity.
Eleven saturation mutagenesis libraries of NBDO were prepared and screened on six sulfides including a drug precursor. Molecular docking and active site volume calculations were used to correlate between the structure of the substrates and the function of the enzymes. The results from this part of the research suggest that the orientation of pro-chiral sulfides in the active site is coordinated by hydrophobic interactions and by steric considerations, which in turn influence the activity and enantioselectivity of NBDO.
In summary, this work sheds light on key residues and on the mechanism of several mono- and dioxygenases, expands the range of pro-chiral substrates and emphasizes the intricacy of relationship between the substrate and the biocatalyst.