Engineering the Regioselectivity of Cyclododecanone Monooxygenase Público

Abstract

Baeyer-Villiger monooxygenases (BVMOs) are the enzymatic alternatives to the chemical Baeyer-Villiger oxidation reaction. The regioselectivity of these enzymes is mostly dependent upon active site residues that govern the stability of the Criegee intermediate and the preferred migratory group of the substrate. The BVMO of interest in this work is cyclododecanone monooxygenase from Rhodococcus ruber SC1 (CDMO). We aim to achieve higher regioselectivity towards two substrates, methyl (2-methyl-6-oxooctan-4-yl) carbamate and 3-methylcyclohexanone. The unnatural product formed as the result of Baeyer-Villiger oxidation with methyl (2-methyl-6-oxooctan-4-yl) carbamate is a -amino acid ester, which after hydrolysis yields useful building blocks for the production of β-peptides, alkaloids, and β-lactam antibiotics. As indicated in literature sources, the conserved arginine-interacting loop in the structure of some BVMOs plays an important role in directing product regioselectivity. We were able to increase the size of the CDMO active site via rational engineering by truncating amino acid side chains on the above-mentioned loop. Here we introduce novel variants with increased activity and regioselectivity towards methyl (2-methyl-6-oxooctan-4-yl) carbamate that also form the unnatural product. Furthermore, we demonstrated that the observed change in regioselectivity does not apply universally, as seen with 3-methylcyclohexanone. Neither of the constructed variants showed promising conversion rates or increased regioselectivity towards 3-methylcyclohexanone and it is potentially due to its small size compared to methyl (2-methyl-6-oxooctan-4-yl) carbamate.    

Table of Contents

1.         Introduction………………………………………....................................….…1

1.1.      Baeyer-Villiger Oxidation…………………….…..............................……..1

1.2.      Baeyer Villiger Monooxygenases (BVMOs)…….......................….….4

1.2.1.   Arginine-Interacting Loop……………………….............................……..6

1.2.2.   Cyclododecanone Monooxygenase………….…..........................………7

2.         Materials and Methods……………………….............................………...11

2.1.      Cloning………………………………………….....................................……...11

2.2.      Expression…………………………………………....................................…..12

2.3.      Purification……………………………….....................................…………….13

2.4.      Activity Assay ………………………....................................…………………14

2.5.      GC Protocol………………………………......................................……………15

2.6.      GCMS Protocol……………………….....................................………………..15

3.         Results and Discussion………………………………..................................16

3.1.      Loop Mutations………………………...................................………………...16

3.2.      Variants P190I/A498G and P190I/L499G……........................….…….21

4.         Conclusion…………………………………………..........................................27

5.         References……………………………………….....................................……...29

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Palabra Clave	Baeyer-Villiger monooxygenases Cyclododecanone monooxygenase
Committee Chair / Thesis Advisor	Lutz, Stefan, Emory University
Committee Members	Weinert, Emily, Emory University Dunham, Christine, Emory University

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