Structure of a eukaryotic thiaminase I

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Thiaminases are enzymes that cleave vitamin B1 (thiamin).
• Thiaminase I enzymes are found in prokaryotes and eukaryotes, with eukaryotic forms typically having higher molecular weights.
• The thiaminase I from the amoeboflagellate Naegleria gruberi is the only characterized eukaryotic thiaminase I.

Purpose of the Study:

• To determine the crystal structure of Naegleria gruberi thiaminase I.
• To elucidate the mechanism of thiamin binding and catalysis by eukaryotic thiaminase I.
• To compare the structure and activity of eukaryotic thiaminase I with its bacterial counterparts.

Main Methods:

• X-ray crystallography was used to solve the structure of N. gruberi thiaminase I at 2.8 Å resolution.
• Isomorphous replacement and pseudo-two-wavelength multiwavelength anomalous diffraction techniques were employed.
• The structure of the enzyme in complex with 3-deazathiamin was determined at 2.7 Å resolution.

Main Results:

• The crystal structure of N. gruberi thiaminase I was determined, revealing structural and activity homology with bacterial thiaminase I.
• The structure in complex with 3-deazathiamin defined the thiamin binding mode and identified Asp272 as the catalytic base.
• The enzyme demonstrated activity with various amines and sulfhydryl compounds as cosubstrates.

Conclusions:

• The crystal structures provide insights into the catalytic mechanism of eukaryotic thiaminase I.
• Despite sequence differences, N. gruberi thiaminase I is structurally and functionally similar to bacterial thiaminase I.
• These findings advance the understanding of thiamin metabolism and enzyme evolution.