Abstract
Thiaminases, enzymes that cleave vitamin B1 into its pyrimidine and thiazole ring moieties, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes accomplish this reaction through substitution of the thiazole ring with a nitrogenous base or sulfhydryl compound. A thiaminase I of the single-celled amoeboflagellate
Naegleria gruberi
is the first eukaryotic thiaminase I to have been examined structurally. The crystal structures in both
apo
form and bound to 3-deazathiamin, a noncleavable thiamin analog and inhibitor of the enzyme, define the mode of thiamin binding to this class of thiaminases and indicate the residues important for catalysis. Comparison with thiaminase II argues for convergent evolution between these two enzymes.
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 (
R
free
, 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;
R
free
, 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.