Does covalent flavin incorporation occur via quinone-methide mechanism in monoamine oxidase?
Betul Kacar and Dale E. Edmondson
Department of Chemistry, Marmara University, Goztepe, Istanbul, Turkey
Department of Chemistry and Biochemistry, Emory University, Atlanta, GA

Abstract

Flavins are yellow chromophores that are found in many organisms ranging from bacteria to humans and function in a variety of biological roles and are essential practically all metabolic processes.(1) Flavins bind to proteins and modulate the catalytic activity of the proteins involved enzymes. One flavoprotein (which is the subject of this project) is “Monoamine Oxidase A”(MAO A). MAO A is a homodimeric flavoenzyme found in the outer mitochondrial membrane of mammals and expressed in a tissue specific manner. MAO A catalyzes the oxidative deamination of neurotransmitters such as serotonin dopamine and norepinephrine. A covalently-bound flavin adenine dinucleotide [FAD] cofactor is present in MAO A in an 8a-thioether linkage to a conserved cysteine residue (Cys-406). The requirement for this covalent FAD attachment and the mechanism of covalent incorporation is not known.(2) The purpose of this study is to investigate the mode of flavin analogue binding test for the proposed Quinone-Methide mechanism. The main goal is to determine the position on the flavin ring that forms the thio ether bond with the enzyme to test the proposed Quinone-Methide mechanism. In this study; 7 8-Diethylriboflavin rather that riboflavin will be used to investigate whether thioether formation occurs at the 8a or 8b-carbon. The proposed Quinone – Methide autocatalytic mechanism predicts specific attachment to the 8a-carbon. Progress to date is the synthesis of 7 8-Diethylriboflavin. Future plans are to biosynthetically incorporate the flavin analog into recombinant MAO A and determine the site of covalent attachment.

Introduction

MAO is a key enzyme of this project which is responsible for the catalyzing the oxidative deamination of the neurotransmitters. It is an outer membrane mitochondrial enzyme existing in two isoforms A and B. MAO A will be studied in order to test the Quinone – Methide mechanism of flavin analogue incorporation. MAO A will be expressed in S. cerevisiae strain that is defficient in riboflavin synthesis. The covalent incorporation of the “Cys-406” (Monoamine Oxidase A) to 7 8-Diethylriboflavin in an 8a position will be an evidence for the auto-catalytic QM mechanism.

Methods and Materials

• Synthesis of 8-ethyl-8-nor-riboflavin
• Biosynthetic incorporation
• Purification and characterization of MAO A with flavin analogue incorporated
• Determination the site of the linkage

Acknowledgements and Funding Attributions

Special thanks to the following Edmondson Lab members for their assistance: Min Li, Milagros Aldeco, and Frantisek Hubalek. This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52003727 and by the National Institute of Health under Grant No. GM-29433.

In Plain English

Enzymes involve many metabolic reactions in the body. They are specific protein molecules which accelarates the life and chemical changes when needed. Monoamine Oxidase A; is a rather specific enzyme that takes part in anti-depressents and Alzheimer and Parkinson disease. They take place with a specific mechanism refer to be Quinone-Methide. Flavin; that is one of the key word of my project is yellow chromophore that is bind to proteins to modulate their catalytic activity. The main goal of the project is to investigate the binding site of the flavin to Monoamine Oxidase that will be an evidence for this auto-catalytic Quinone-Methide mechanism.

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