The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution

Abstract

This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.

Mutagenic and carcinogenic structural alerts and their mechanisms of action

“…Acylation is a reaction where reactive tetrahedral intermediate is formed with a nucleophile, and the leaving group is released. Schiff base formation is a mechanism where electrophilic carbon atoms of aldehydes or ketones are attacked by amines and the C=O double bond is replaced by the C=N double bond (32). Michael addition is a two-step nucleophilic addition, where nucleophile attacks a double bond and forms two new bonds.…”

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

“…The most abundant cyst wall glycoprotein (Jacob), which was acidic and was 100 kDa, was excised from a Coomassie blue-stained gel and digested with trypsin. Tryptic peptides were separated by high-pressure liquid chromatography, and N-terminal sequences were obtained by Edman degradation (32). Alternatively, two-dimensional protein gels of cyst wall proteins were transferred to polyvinylidene difluoride (PVDF) filters, and Jacob, identified with Ponceau-S., was excised for N-terminal sequencing.…”

Enforced Presentation of an Extrahelical Guanine to the Lesion Recognition Pocket of Human 8-Oxoguanine Glycosylase, hOGG1

“…In previous studies, we determined that oxoG excision by hOGG1 proceeds via a covalent catalysis mechanism in which the oxoG is subject to nucleophilic displacement by the ⑀-amino group of Lys-249 (10,11). Mutation of this residue to the non-nucleophilic Gln generates a variant of the enzyme (K249Q hOGG1) that lacks catalytic activity but retains the ability to recognize oxoG-containing DNA specifically.…”