Cloning, Heterologous Expression, and Characterization of Human and Escherichia Coli Choline Dehydrogenases
Gratson, Alejandra Araya
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https://hdl.handle.net/2142/84944
Description
Title
Cloning, Heterologous Expression, and Characterization of Human and Escherichia Coli Choline Dehydrogenases
Author(s)
Gratson, Alejandra Araya
Issue Date
2005
Doctoral Committee Chair(s)
Timothy Garrow
Department of Study
Nutritional Sciences
Discipline
Nutritional Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Membrane bound choline dehydrogenase (CHDH) catalyzes the first committed step of choline oxidation to betaine-glycine in mammals and in some bacteria such as E. coli and P. aeruginosa . In mammals, it is an important source of one-carbon units for cytosolic folate- and AdoMet-dependent reactions, while in E. coli, betaine-glycine, the final product of choline oxidation, acts as an osmopretectant compatible solute that accumulates when the cells are exposed to drastic environmental changes in osmolarity. Betaine-glycine also plays an important role in osmoprotection in mammalian cells exposed to high extracellular osmolarity, such as kidney cells. Since CHDH is the first committed step of choline oxidation, it may be a critical regulatory enzyme governing flux through this pathway. Mammalian CHDH has been studied most extensively in rat liver, but difficulties in obtaining a highly purified enzyme preparation have hampered further studies with this enzyme. In E. coli, the biosynthetic pathway for the production of betaine-glycine from choline has been well characterized at the genetic level, but only minimal biochemical studies on CHDH have been reported to date. Although, based on primary sequence alignment, the enzyme has been grouped in the glucose-methanol-choline (GMC) flavin-dependent oxidoreductase superfamily, no clear biochemical evidence has been shown indicating the presence of flavin adenine dinucleotide (FAD) as a cofactor. This electron transport chain linked enzyme shows an absolute requirement for an electron acceptor other than molecular oxygen, which has been proposed to be Coenzyme Q10 in mammals, based on studies performed with rat liver mitochondria and partially purified enzyme. As a first step towards a biochemical and mechanistic characterization of CHDH, both human and E. coli CHDH have been cloned and expressed in E. coli and partially purified to homogeneity. Here, using UV-visible spectroscopical analysis and thin layer chromatography, FAD has been identified as the cofactor for purified recombinant E. coli CHDH, which is also shown to use coenzyme Q1 as its electron acceptor. The affinity of purified E. coli CHDH towards its substrates, choline and coenzyme Q1, is also reported.
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