Mechanistic Studies on the Radical SAM Enzyme Tryptophan Lyase (NosL).
| Autor: | Bhandari DM; Department of Chemistry, Texas A&M University, College Station, TX, United States., Fedoseyenko D; Department of Chemistry, Texas A&M University, College Station, TX, United States., Begley TP; Department of Chemistry, Texas A&M University, College Station, TX, United States. Electronic address: begley@tamu.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in enzymology [Methods Enzymol] 2018; Vol. 606, pp. 155-178. Date of Electronic Publication: 2018 Jul 19. |
| DOI: | 10.1016/bs.mie.2018.06.008 |
| Abstrakt: | Tryptophan lyase (NosL) is a radical SAM enzyme that catalyzes the formation of 3-methyl-2-indolic acid from l-tryptophan in the biosynthesis of the antibiotic nosiheptide. NosL is the newest addition to the radical SAM-dependent aromatic amino acid lyase subfamily which includes ThiH, HydG, and CofH. The recently solved crystal structure of NosL challenged the previously accepted mechanistic hypothesis and spurred a renewed interest in investigating the reaction. This led to a series of studies that unraveled several fascinating aspects of the fragmentation-recombination reaction. This chapter describes the various methodologies used for the overexpression of NosL, its purification, in vitro reconstitution, preparation of isotopically labeled substrates, and chemoenzymatic synthesis of substrate analogs. The methods described here can be used to further investigate other aromatic amino acid lyases as well as reactivity of fleeting radicals in enzymology. (© 2018 Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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