Crystallization studies of 5'-deoxyadenosyl radical enzymes

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Phillips, Laura (Laura Anne)

Advisor dc:contributor.advisor

• Catherine L. Drennan.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/41769

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/41769