Structural Characterization of the Regulatory Proteins TenA and TenI from Bacillus subtilis and Identification of TenA as a Thiaminase II^,

Abstract

Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.

Both Thiamine Uptake and Biosynthesis of Thiamine Precursors Are Required for Intracellular Replication ofListeria monocytogenes

“…As alternative explanation, the same authors proposed the existence of unidentified HMP and HET transporters or the erroneous assignment of thiamine specificity to Lmo1429 homologs. lmo0315 within the same listerial operon encodes thiaminase II (TenA), which is involved in thiamine hydrolysis and/or the regeneration of the thiamine precursor HMP (14,34).…”

Structure of a eukaryotic thiaminase I

“…The Ngthiaminase structure suggests that the eukaryotic enzymes can exist as a single polypeptide, although perhaps sensitive to proteolytic modification, rather than native aggregates of smaller polypeptide subunits as observed for thiaminase II enzymes like TenA (49). It is interesting to note that Saccharomyces cerevisiae possesses multifunctional enzymes involved in thiamin salvage and biosynthesis.…”

“…S6) (49). Although this enzyme has a completely different fold from that of thiaminase I, the pyrimidine of HMP makes analogous interactions with the same types of residues in TenA as does 3-dzThi with Ng-thiaminase, including N1′ hydrogen bonding to Glu205 (Glu232), N3′ hydrogen bonding to Asp44 (Asp262), and N4′ hydrogen bonding to Asp44 and Tyr163 (Asp262 and Tyr53) (49). Because both thiaminase I and thiaminase II catalyze the same reaction, the only difference being the type of nucleophile used, one could argue for convergent evolution between these two enzymes.…”

Origins for Micronutrient Deficiencies

“…There are multiple studies of the effect of bacteria on vitamin B12 and thiamine. A major observation in this field has been that bacteria secrete thiaminases, which can cleave thiamine (61)(62)(63). Staphylococcus aureus produces a thiaminase type II that is regulated at both the transcriptional as well as the enzymatic level.…”