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The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria - PubMed

️Mon Jan 01 2024

The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria

Kaede Kachi et al. Appl Environ Microbiol. 2024.

Abstract

Tetrahydrofolate is a cofactor involved in C₁ metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria Lactococcus lactis and Escherichia coli, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium Limosilactobacillus reuteri. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the lreu_1276 gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using E. coli and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg²⁺, the Lreu_1276 protein exhibited maximum activity in the presence of Mn²⁺, with a specific activity of 28.2 ± 2.0 µmol min^-1 mg^-1 in the presence of 1 mM Mn²⁺. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria.

Importance: The identification of a structurally novel DHNTP pyrophosphohydrolase in L. reuteri provides valuable information in understanding tetrahydrofolate biosynthesis in bacteria that possess lreu_1276 homologs. Interestingly, however, even with the identification of a third family of DHNTP pyrophosphohydrolases, there are still a number of bacteria that do not harbor homologs for any of the three genes while possessing other genes involved in the biosynthesis of the pterin ring structure. This suggests the presence of an unrecognized DHNTP pyrophosphohydrolase gene in bacteria. As humans do not harbor DHNTP pyrophosphohydrolase, the high structural diversity of enzymes responsible for a reaction in tetrahydrofolate biosynthesis may provide an advantage in designing inhibitors targeting a specific group of bacteria in the intestinal microbiota.

Keywords: Limosilactobacillus; biosynthesis; dihydroneopterin triphosphate pyrophosphohydrolase; folate; intestinal bacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Fig 1**
The predicted tetrahydrofolate biosynthesis pathway in *L. reuteri*. Gene locus tags are shown in parenthesis. Red broken arrows indicate reactions catalyzed by enzymes that have not been identified in *L. reuteri*. Abbreviations: DHNTP, 7,8-dihydroneopterin triphosphate; DHN, 7,8-dihydroneopterin; 6-HMD, 6-hydroxymethyl-7,8-dihydropterin; DHPPP, 6-hydroxymethyl-7,8-dihydropterin diphosphate; DHP, 7,8-dihydropteorate.

**Fig 2**
Distribution of genes involved in dihydrofolate biosynthesis. Black and white circles indicate the presence and absence of gene homolog, respectively. In *folEBKP* column, gray circles with *EKP* and *BKP* show the presence of *folEKP* and *folBKP* gene homologs, respectively. G-T indicates a set of GCH-II and TrpF_CtL2. Proteins displaying e-values less than 1 × 10e-5, 6 × 10e-25, and 2 × 10e-17 toward PTPS-III (CLC_0882), GCH-II (CT_731), and TrpF_CtL2 (Ctl0581) were recognized as their homologs, respectively. The PTPS-III protein and GCH-II/TrpF_CtL2 proteins can form pathways that bypass those composed of FolQB and FolEQ proteins, respectively. Red letters indicate organisms harboring homologs of the *folQ3* gene identified in this study, whereas blue letters indicate organisms without any already-identified *folQ* genes, although they possess other genes predicted to be involved in dihydrofolate biosynthesis (*folE*, *folB*, *folK*, and *folP*) and do not possess possible bypass pathways. †Organisms with GTP cyclohydrolase IB homolog instead of FolE homolog. *Organisms harboring a set of FolE, FolB, FolK, and FolP homologs, although similarity between some of the proteins and corresponding query proteins is lower than the threshold in Table S1.

**Fig 3**
Schematic diagram of the predicted dihydrofolate biosynthesis operon in *L. reuteri*. Arrowed boxes indicate genes in the predicted dihydrofolate biosynthesis gene cluster on the *L. reuteri* genome. *lreu_1275* and *lreu_1277-lreu_1280* genes are predicted to encode proteins involved in dihydrofolate biosynthesis shown in Fig. 1. The *lreu_1276* gene, forming an operon with these genes, is annotated as Ham1 family protein in GenBank and as XTP/dITP diphosphohydrolase in KEGG database and its function is unclear.

**Fig 4**
Purified Ec-FolE and Lreu_1276 recombinant proteins. One microgram of Ec-FolE (A) and 4 μg of Lreu_1276 (B) recombinant proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie Brilliant Blue. M indicates molecular mass marker.

**Fig 5**
The HPLC and LC-MS analyses of Ec-FolE reaction products. (A) HPLC analysis was carried out toward reaction products without and with Ec-FolE enzyme indicated with blue and red lines, respectively. LC-MS analysis was performed toward reaction products without (B) and with the enzyme (C).

**Fig 6**
The HPLC and LC-MS analyses of Lreu_1276 reaction products. (A) HPLC analysis was carried out toward Lreu_1276 reaction product. Black, blue, and red lines indicate the DHNMP standard compound, reaction product without the enzyme, and reaction product with the enzyme, respectively. LC-MS analysis was performed toward reaction products without (B) and with the enzyme (C).

**Fig 7**
Metal ion dependency of DHNTP pyrophosphohydrolase reaction catalyzed by the Lreu_1276 recombinant protein. (A) DHNTP pyrophosphohydrolase activity was measured in the presence of various metal ions (1 mM each). For examination of Mn²⁺, Mg²⁺, and Zn²⁺ ions, 100 mM DHNTP was used as a substrate, whereas for other ions, 150 mM DHNTP was used as a substrate. (B) DHNTP pyrophosphohydrolase activity was measured in the presence of various concentrations of Mn²⁺ ions. Error bars indicate the standard deviations of three independent experiments.

**Fig 8**
Enzymatic properties of Lreu_1276 recombinant protein. (A) Effects of reaction temperature on pyrophosphohydrolase activity of Lreu_1276 protein were examined. (B) Effects of pH on pyrophosphohydrolase activity were investigated. Error bars represent the standard deviations of three independent experiments.

**Fig 9**
Structural analysis of Ll-FolQ1, Ec-FolQ2, and Lr-FolQ3 proteins. The structures of DHNTP pyrophosphohydrolases from *L. reuteri* (Lr-FolQ3) (A) and *L. lactis* (Ll-FolQ1) (B) predicted by AlphaFold2 and deposited in AlphaFold Protein Structure Database (AF-A5VL09-F1-model_v4 and AF-P0CI35-F1-model_v4, respectively) were utilized. Structure of the enzyme from *E. coli* (monomeric form Ec-FolQ2), which has been published in PDB database (2O5W), was also utilized (C). Structures of Ll-FolQ1 (magenta) and Ec-FolQ2 (green) (D), Lr-FolQ3 (cyan) and Ll-FolQ1 (magenta) (E), and Lr-FolQ3 (cyan) and Ec-FolQ2 (green) (F) were superimposed. Superimposition was performed utilizing PyMOL software with a cutoff of 2.0.

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The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria - PubMed