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Thursday, October 10, 2013

A Radical Transfer Pathway in Spore Photoproduct Lyase

Chemical Biology Chembio Seminar
1640 Chemistry
4:00 PM - 5:30 PM
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Contact:   nmarsh@umich.edu

Speaker:   Lei Li, Indiana University-Purdue University Indianapolis
Abstract:   The dominant DNA photo-damage product detected in UV irradiated bacterial endospores is a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP. SP accumulates in dormant endospores and is repaired rapidly by a metalloenzyme named spore photoproduct lyase (SPL) at the bacterial early germination phase. SPL utilizes a unique [4Fe-4S] cluster coupled by S-adenosylmethionine (SAM) to generate a 5′-deoxyadenosyl radical to repair SP and belongs to the radical SAM superfamily. By utilizing the deuterium labeled SP substrates, we proved that SPL initiates the SP repair process by abstracting the H6proR atom. The resulting SP C6 radical fragments to yield a thymine methyl radical, which subsequently abstracts an H atom from a protein residue to produce the repaired thymine. The H-donating residue is implied to be the cysteine 141 in B. subtilis SPL, as demonstrated by cysteine labeling experiments, enzyme kinetic data, as well as protein crystallographic studies. Such a mechanism results in a thiyl radical on this cysteine and this radical must be involved in the subsequent SAM regeneration process. Our latest data show that two tyrosine residues are also involved in catalysis, suggesting that SPL harbors a novel radical transfer pathway which is composed by at least four hydrogen atom transfer (HAT) processes and is responsible for SAM regeneration at the end of each catalytic cycle. This is the first radical transfer pathway found in the radical SAM superfamily (> 44,000 members). It partially resembles the radical transfer chain in the R1 subunit of the class I ribonucleotide reductase and is of significance to our understanding of the radical relay process in enzyme catalysis.