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theses

Several enzymes use more than just energy to perform catalysis. Reactions requiring harsh conditions and energy requirement that exceed the cell’s overall energy output are made possible by enzymes. Several of these enzymes are known for using cofactors in their catalysis. One such class of enzyme known as, “thiamin diphosphate dependent enzyme” uses thiamin diphosphate (ThDP) an active form of vitamin B1 to carry out reactions that include cleavage of carbon-carbon, carbon-sulfur, carbon-oxygen and carbon-nitrogen bonds. The understanding of its mechanism has opened a new spectrum of green chemistry involving manipulation of enzymes to give side reactions which create chemical bonds instead of breaking them. Herein, two different ThDP-dependent enzymes and their carboligation reactivity was studied. E1o which is a component of the oxoglutarate dehydrogenase multienzyme complex (OGDHc) and MenD, an enzyme involved in the biosynthesis reaction of vitamin K2. MenD and E1o’s ability to carry out carboligation reaction of bulky aromatic and short aliphatic acceptors with substrate was studied using circular dichroism (CD). The observations using CD were made possible due to an inherent property of the activated nucleophile, “prochirality.” The reaction between the prochiral nucleophile and electrophile led to the formation of chiral products which in turn were observed. To carry out these reactions the plasmid carrying enzyme genes were first over-expressed in E. coli cells and purified using IMAC chromatography (immobilized metal affinity chromatography). The enzymatic reactions were carried out using 2-oxoglutarate (substrate), benzaldehyde (acceptor) and propanal (acceptor) giving their respective products 5-hydroxy-4-oxo-5-phenylpentanoic acid and 5-hydroxy-4-oxo-heptanoic acid. The reactions were also performed using a different 2-oxo-acid (substrate), 2-oxo-5-hexenoic acid with benzaldehyde (acceptor) and propanal (acceptor). The results showed, MenD’s activity was limited due to its mechanism at high substrate levels, leading to little to no product formation attributed to its ping-pong bi-bi mechanism. Moreover, MenD despite having very little sequence similarity to E1o follows a similar reaction mechanism. This opens new opportunities for MenD’s substrate and acceptor spectrum, and its saturation inhibition issues which can be addressed by knocking out or substituting part of its active site residues, or the hydrophobic chains supporting it, using site-directed mutagenesis.

ETD_8936

M.S.

Includes bibliographical references

by Abhilash S. Christian

doi:10.7282/T36Q21NW

ETD graduate

The author owns the copyright to this work.