Structural and mutational analysis of TenA protein (HP1287) from the Helicobacter pylori thiamin salvage pathway – evidence of a different substrate specificity

Abstract

HP1287 (tenA) from Helicobacter pylori is included among the genes that play a relevant role in bacterium colonization and persistence. The gene has been cloned and its product, protein TenA, has been expressed and purified. The crystal structures of the wild‐type protein and the mutant F47Y have been determined at resolutions of 2.7 and 2.4 Å, respectively. The molecular model, a homotetramer with 222 symmetry, shows that the H. pylori TenA structure belongs to the thiaminase II class of proteins. These enzymes were recently found to be involved in a salvage pathway for the synthesis of the thiamin precursor hydroxypyrimidine, which constitutes a building block in thiamin biosynthesis, in particular in bacteria living in the soil. By contrast, enzymatic measurements on TenA from H. pylori indicate that the activity on the putative substrate 4‐amino‐5‐aminomethyl‐2‐methylpyrimidine is very modest. Moreover, in the present study, we demonstrate that the mutation at residue 47, a position where a phenylalanine occurs in all the strains of H. pylori sequenced to date, is not sufficient to explain the very low catalytic activity toward the expected substrate. As a result of differences in the colonization environment of H. pylori as well as the TenA structural and catalytic peculiar features, we suggest a possible pivotal role for the H. pylori enzyme in the thiamin biosynthetic route, which is in agreement with the relevance of this protein in the stomach colonization process. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260232: TenA (uniprotkb:http://www.ebi.uniprot.org/entry/O25874) and TenA (uniprotkb:http://www.ebi.uniprot.org/entry/O25874) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by x‐ray crystallography (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114)

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

“…We detected activity only against amino-HMP ( Figure 5B). This activity was quite low, 5.4 nmol min 21 mg 21 protein, i.e., one to two orders of magnitude less than standalone bacterial TenA_C proteins (Jenkins et al, 2007;Barison et al, 2009). Mutating the active-site cysteine (Cys-213) to alanine abolished the activity ( Figure 5C), confirming that it resides in the TenA domain.…”

“…That this activity was low could be due to instability during isolation because activity appeared not to survive beyond the Ni 2+ -affinity step in purification. The low activity could also be because amino-HMP is not the physiological substrate, this substrate being a breakdown product upstream of amino-HMP ( Figure 5A), as proposed for Helicobacter pylori TenA_C (Barison et al, 2009). If so, the TenA-HAD fusion could be rationalized as follows.…”

“…TenA_C proteins from yeast (Onozuka et al, 2008) and bacteria (Jenkins et al, 2007;Barison et al, 2009) participate in salvage of the thiamin pyrimidine moiety; they hydrolyze the breakdown product 4-amino-5-aminomethyl-2-methylpyrimidine (amino-HMP) to the thiamin precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) ( Figure 5A) and can in some cases also slowly hydrolyze thiamin itself. TenA_C proteins lack activity against the N-formyl derivative of amino-HMP (another thiamin breakdown product; Figure 5A) (Jenkins et al, 2007;Zallot et al, 2014).…”

In silico analysis of heme oxygenase structural homologues identifies group‐specific conservations

“…Interestingly, a Y47F mutation at index 183 in BsThiaminase leads to a significant impairment of catalytic function [21]. Also, a F47Y mutation at index 183 in Helicobacter pylori TenA disrupts the helical folding of helices D and E [43].…”

“…The pyrimidine ring of HMP-P is packed between the side chains of Phe46{183} and Tyr132{332} in PfThiaminase [17]. Mutations at this index position in BsThiaminase and H. pylori TenA thiaminase decrease catalytic activity and disrupt helical folding [21,43]. In MMOHs, Gly113{183} in the alpha chain lines the pocket for the diiron cluster [16].…”