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TitleStructure of a eukaryotic thiaminase I.
Publication TypeJournal Article
Year of Publication2014
AuthorsKreinbring CA, Remillard SP, Hubbard P, Brodkin HR, Leeper FJ, Hawksley D, Lai EY, Fulton C, Petsko GA, Ringe D
JournalProc Natl Acad Sci U S A
Volume111
Issue1
Pagination137-42
Date Published2014 Jan 7
ISSN1091-6490
KeywordsCatalysis, Catalytic Domain, Crystallography, X-Ray, Escherichia coli, Hydrolases, Mercaptoethanol, Naegleria, Peptides, Protein Binding, Thiamine
Abstract

Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed. Here, we present the crystal structure of N. gruberi thiaminase I to a resolution of 2.8 Å, solved by isomorphous replacement and pseudo-two-wavelength multiwavelength anomalous diffraction and refined to an R factor of 0.231 (Rfree, 0.265). This structure was used to solve the structure of the enzyme in complex with 3-deazathiamin, a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; Rfree, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as l-cysteine and β-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.

DOI10.1073/pnas.1315882110
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID24351929
PubMed Central IDPMC3890816
Grant ListP41 RR007707 / RR / NCRR NIH HHS / United States
R24 GM111072 / GM / NIGMS NIH HHS / United States
Y1-CO-1020 / CO / NCI NIH HHS / United States
Y1-GM-1104 / GM / NIGMS NIH HHS / United States