Crystallization studies of 5'-deoxyadenosyl radical enzymes
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226297726-MIT.pdf
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Author(s)
Phillips, Laura (Laura Anne)
Advisor(s)
Catherine L. Drennan.
Date Issued
2007
Publisher
Massachusetts Institute of Technology
Abstract
Both adenosylcobalamin- and S-adenosylmethionine-dependent radical enzymes use a 5'-deoxyadenosyl radical intermediate to abstract a hydrogen atom from their substrates. In the case of adenosylcobalamin-dependent enzymes, the 5'-deoxyadenosyl radical is generated by homolytic cleavage of the carbon-cobalt bond of adenosylcobalamin. In the case of S-adenosylmethionine-dependent radical enzymes, the 5'-deoxyadenosyl radical is generated by reductive cleavage of the S-adenosylmethionine following injection of an electron into the sulfur atom by a reduced [4Fe-4S] cluster. Most known structures of adenosylcobalamin- and S-adenosylmethionine-dependent radical enzymes show that the enzyme active site is in a full or partial TIM barrel. In order to further understanding of the catalytic requirements of enzymes in these classes, crystallization studies were undertaken on four enzymes. The structure of the resting form of lysine 5,6-aminomutase, an adenosylcobalamin-dependent enzyme, is known from previous work in our laboratory; however, the structure of a catalytic state has not been solved. Here, crystallization experiments were performed to try to trap the catalytic enzyme form. Human adenosyltransferase catalyzes the formation of adenosylcobalamin from cob(II)alamin and adenosine triphosphate. Crystallization experiments were set up with and without cobalamin to try to solve its structure. Lipoate synthase is another Sadenosylmethionine-dependent radical enzyme, performing two sulfur insertion reactions on a protein-bound octanoyl group to form a lipoyl group. Crystallization experiments were performed on this enzyme, with and without the substrate, in an attempt to solve its structure and better understand the mechanism of sulfur insertion.
(cont.) Class III ribonucleotide reductase activase is an S-adenosylmethionine-dependent radical enzyme that activates class III ribonucleotide reductase by abstracting a hydrogen atom from a glycine residue. Crystallization experiments were performed with both enzymes present in order to try to determine the structure of the complex; the activase is unstable alone. Thus far, none of the crystallization experiments described here resulted in crystals of high enough quality to solve the structures of these enzymes.
Description
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007.
Vita.
Includes bibliographical references.
Subjects
Chemistry.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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