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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Catalysis of a flavoenzyme-mediated amide hydrolysis
Tathagata Mukherjee1, Yang Zhang, Sameh Abdelwahed
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Journal of the American Chemical Society
|April 8, 2010
Summary
Researchers discovered a novel flavoenzyme, RutA, that initiates the pyrimidine catabolic pathway in E. coli by oxidizing uracil. This finding reveals a unique oxidative mechanism for ring opening, highlighting the flavin cofactor
Area of Science:
- Biochemistry
- Enzymology
- Microbial Metabolism
Background:
- A novel pyrimidine catabolic pathway, the Rut pathway, has been identified in Escherichia coli K12.
- This pathway converts uracil into 3-hydroxypropionate, ammonia, and carbon dioxide, requiring the seven-gene Rut operon.
Purpose of the Study:
- To identify the enzyme catalyzing the initial uracil ring-opening reaction in the Rut pathway.
- To elucidate the mechanism of this initial reaction catalyzed by the flavoenzyme RutA.
Main Methods:
- Enzymatic assays using the flavoenzyme RutA.
- Mechanistic studies to determine the reaction pathway.
- Characterization of reaction intermediates and products.
Main Results:
- The flavoenzyme RutA was demonstrated to catalyze the initial uracil ring-opening reaction, producing 3-ureidoacrylate.
- The reaction proceeds via an oxidative mechanism involving flavin hydroperoxide addition to the uracil C4 carbonyl.
- This represents a novel example of flavin hydroperoxide-mediated chemistry in amide hydrolysis.
Conclusions:
- RutA is the enzyme responsible for the initial step in the Escherichia coli pyrimidine catabolic pathway.
- The study reveals an unprecedented oxidative mechanism for uracil ring opening by a flavoenzyme.
- This work underscores the catalytic diversity and adaptability of the flavin cofactor in biological systems.
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