טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRoi Feingersch
SubjectProtein Engineering of Toluene Monooxygenases for Synthesis
of Chiral Sulfoxides
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Fishman Ayelet
Full Thesis textFull thesis text - English Version


Abstract

The use of enzymes for the synthesis of chiral drugs is well established in recent years, and biotransformations are now accepted as a common methodology for the preparation of chiral pharmaceuticals.  Enantiopure sulfoxides are valuable asymmetric starting materials that are able to bring about asymmetric transformations and are important chiral auxiliaries in organic synthesis.  Additionally,  they possess a wide range of biological activities.  It was the goal of this research to prepare chiral sulfoxides via asymmetric oxidation of sulfides using toluene monooxygenases (TMOs).

TMOs are soluble, non-heme diiron multi-component enzymes which utilize oxygen to catalyze the initial hydroxylation step in metabolic pathways for the oxidation of hydrocarbon substrates. TMOs were shown to be versatile biocatalysts capable of oxidizing a large spectrum of substrates such as substituted aromatic and phenolic compounds. Here we show for the first time, that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides.  Furthermore, through mutagenesis of a key position in the α-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, the catalytic activity is altered so that the rate and enantioselectivity are dramatically improved. TOM variant TomA3 V106M was found to hydroxylate methyl-phenyl-sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme. The enantiomeric excess (pro-S) increased from 51% for the wild-type to 88% for the mutant. T4MO variant TmoA I100G increased the wild-type oxidation rate by 1.73-fold and the enantiomeric excess rose from 86% to 98% (pro-S). TOM variant V106A oxidized methyl para-tolyl sulfide to the corresponding sulfoxide at a rate of 2.0 nmol/min/mg protein which was 4.0 times higher than the wild-type, while the enantiomeric excess increased from nearly racemic to 50%. Even more remarkable results were obtained with T4MO TmoA I100G, which oxidized methyl para-tolyl sulfide 11.4 times faster than wild-type and changed the selectivity from 41% pro-R to 77% pro-S. A correlation between regioselectivity and enantioselectivity was shown for TMOs studied in this work.

In silico modeling was used to elucidate some structure-function relationships of the substrates and mutants.  It was shown, that position V106 of TOM (and I100 of T4MO), controls the size of the hydrophobic gate thus restricting the size of entering substrates and influencing the reaction rate.  Additionally, this position governs proper alignment of the substrate near the diiron atoms thus controlling the enantioselectivity of the product.