Aromatic Amino Acid Residues Research Articles

Structure of the sensory domain of McpX from Sinorhizobium meliloti, the first known bacterial chemotactic sensor for quaternary ammonium compounds.

The α-proteobacterium Sinorhizobium meliloti can live freely in the soil or engage in a symbiosis with its legume host. S. meliloti facilitates nitrogen fixation in root nodules, thus providing pivotal, utilizable nitrogen to the host. The organism has eight chemoreceptors, namely McpT to McpZ and IcpA that facilitate chemotaxis. McpX is the first known bacterial sensor of quaternary ammonium compounds (QACs) such as choline and betaines. Because QACs are exuded at chemotaxis-relevant concentrations by germinating alfalfa seeds, McpX has been proposed to contribute to host-specific chemotaxis. We have determined the crystal structure of the McpX periplasmic region (McpXPR) in complex with the proline betaine at 2.7 Å resolution. In the crystal, the protein forms a symmetric dimer with one proline betaine molecule bound to each monomer of McpXPR within membrane-distal CACHE module. The ligand is bound through cation-πinteractions with four aromatic amino acid residues. Mutational analysis in conjunction with binding studies revealed that a conserved aspartate residue is pivotal for ligand binding. We discovered that, in a striking example of convergent evolution, the ligand-binding site of McpXPR resembles that of a group of structurally unrelated betaine-binding proteins including ProX and OpuAC. Through this comparison and docking studies, we rationalized the specificity of McpXPR for this specific group of ligands. Collectively, our structural, biochemical, and molecular docking data have revealed the molecular determinants in McpX that are crucial for its rare ligand specificity for QACs.

Starch Synthesis in Ostreococcus tauri: The Starch-Binding Domains of Starch Synthase III-B Are Essential for Catalytic Activity.

Starch is the major energy storage carbohydrate in photosynthetic eukaryotes. Several enzymes are involved in building highly organized semi-crystalline starch granules, including starch-synthase III (SSIII), which is widely conserved in photosynthetic organisms. This enzyme catalyzes the extension of the α-1,4 glucan chain and plays a regulatory role in the synthesis of starch. Interestingly, unlike most plants, the unicellular green alga Ostreococcus tauri has three SSIII isoforms. In the present study, we describe the structure and function of OsttaSSIII-B, which has a similar modular organization to SSIII in higher plants, comprising three putative starch-binding domains (SBDs) at the N-terminal region and a C-terminal catalytic domain (CD). Purified recombinant OsttaSSIII-B displayed a high affinity toward branched polysaccharides such as glycogen and amylopectin, and to ADP-glucose. Lower catalytic activity was detected for the CD lacking the associated SBDs, suggesting that they are necessary for enzyme function. Moreover, analysis of enzyme kinetic and polysaccharide-binding parameters of site-directed mutants with modified conserved aromatic amino acid residues W122, Y124, F138, Y147, W279, and W304, belonging to the SBDs, revealed their importance for polysaccharide binding and SS activity. Our results suggest that OT_ostta13g01200 encodes a functional SSIII comprising three SBD domains that are critical for enzyme function.

Role of (single/double chain surfactant) micelles on the protein aggregation

To investigate the interaction between the bovine serum albumin (BSA) and cationic surfactants (monomeric, cetyltrimethylammonium bromide, CTAB) and dimeric/gemini, 1, 6 bis (N, N-hexadecyl dimethyl ammonium bromide, 16-6-16) and to find out the role of micelles in the aggregation of the protein using spectroscopic (UV–visible, fluorescence, fluorescence lifetime measurements, circular dichroism (CD), etc.) and microscopic (atomic force microscope (AFM)) techniques. The different surfactant has an effect on the polarity of the microenvironment of the protein shows in all the spectroscopic technique at below and above the critical micelle concentration (CMC). The far-UV CD spectra show that BSA is more disrupted by the dimeric surfactant compared to the monomeric CTAB above the CMC. The binding of the surfactant induce changes in the microenvironment around the aromatic amino acids residues and disulfide bond of the BSA at different pHs. The binding constant values were found to be 20.278×103M and 8.443M for the BSA-CTAB complex and BSA-16-6-16 complex, respectively. Atomic force microscope indicates the aggregation is more in case of dimeric (16-6-16) surfactant compared to the monomeric (CTAB) surfactant at the higher concentration (above their CMCs). Below and above the CMC, all changes are noticeable.

Use of Microscale Thermophoresis (MST) to Measure Binding Affinities of Components of the Fusion Machinery.

Microscale thermophoresis is a relatively new technique used by an increasing number of academic laboratories to estimate relative binding affinities between ligand (analyte) that is titrated and a target (generally protein) that is either fluorescently labeled exogenously in the red or blue channel (labeled thermophoresis) or endogenously labeled via the presence of sufficient aromatic amino acid residues such as tryptophan (label-free thermophoresis). There are advantages and disadvantages to each technique; however, one major disadvantage of label-free thermophoresis is that protein-protein interactions cannot be measured, as generally most proteins have enough aromatic residues to generate an interfering signal. Thermophoresis can be used to determine steady-state binding affinities as between SNAREs and relevant binding partners of SNAREs and labeled thermophoresis is increasingly becoming a reliable technique to screen binding partners of fusion machinery to determine relevance in terms of direct biochemical interactions.

On-Axis Alignment of Protein Nanocage Assemblies from 2D to 3D through the Aromatic Stacking Interactions of Amino Acid Residues.

Aromatic-aromatic interactions between natural aromatic amino acids Phe, Tyr, and Trp play crucial roles in protein-protein recognition and protein folding. However, the function of such interactions in the preparation of different dimensional, ordered protein superstructures has not been recognized. Herein, by a combination of the directionality of the symmetry axes of protein building blocks and the strength of the aromatic-aromatic interactions coming from a group of aromatic amino acid residues, we built an engineering strategy to construct protein superlattices. Based on this strategy, substitution of single amino acid residue Glu162 around the C4 rotation axes near the outer surface of 24-mer ferritin nanocage with Phe, Tyr, and Trp, respectively, resulted in 2D and 3D protein superlattices where protein cages are aligned along the C4 axes, imposing a fixed disposition of neighboring ferritins. The self-assembly of these superlattices is reversible, which can be tuned by external stimuli (salt concentration or pH). Moreover, these superlattices can serve as biotemplates for the fabrication of 2D and 3D inorganic nanoparticle arrays.

Molecular interactions, bioavailability, and cellular mechanisms of angiotensin-converting enzyme inhibitory peptides.

Food-derived angiotensin-converting enzyme inhibitory (ACEi) peptides have gained substantial interest as potential alternatives to synthetic drugs in the management of hypertension. Peptide size and sequence are two critical factors that determine their potency, bioavailability, and cellular mechanisms. Molecular interaction studies between ACE and ACEi peptides support that potent ACEi peptides are generally composed of hydrophobic, positively charged, and aromatic or cyclic amino acid residues at the third, second, and first position from the C-terminus, respectively. Small peptides containing N-terminal Tyr and/or C-terminal Pro could improve their stability against enterocyte peptidases, thus their bioavailability. Different ACEi peptides can reduce aberrant cellular proliferation, excessive inflammation, and oxidative stress but through different mechanisms. Further understanding the structure-activity-bioavailability relationships will help design novel potent ACEi peptides with improved bioavailability and in vivo efficacy. PRACTICAL APPLICATIONS: ACEi peptides have the potential for uses as functional food ingredients against hypertension.

Inhibition of the Exoglucanase Cel7A by a Douglas-Fir-Condensed Tannin.

Douglas-fir forestry residues are a potential feedstock for saccharification-based biofuels, and condensed tannins are expected to make up ∼3% of the dry mass of this feedstock. Condensed tannins are well-known for their ability to interact with proteins and can bind and inhibit cellulase enzymes used in saccharification. In this study, we use molecular docking and classical molecular dynamics simulations to investigate how a characterized condensed tannin from Douglas-fir bark binds to the exoglucanase Cel7A from Trichoderma reesei. Through looking at the "occupancy" and "residency" of specific amino acid residue-tannin interactions, we find that the binding sites are characterized by many simultaneous tannin-enzyme interactions with the strongest occurring on the catalytic module as opposed to the carbohydrate-binding module. The simulations indicate that tannin inhibition can result from binding at or near the catalytic tunnel's entrance and exit. The analyzed tannin further prefers to bind to loops around the catalytic region and has affinity for aromatic and charged amino acid residues. These insights provide direction for the rational design of tannin-resistant cellulases.

Isoform selectivity of harmine-conjugated 1,2,3-triazoles against human monoamine oxidase.

There is little information available on the monoamine oxidase isoform selectivity of N-alkyl harmine analogs, which exhibit a myriad of activities including MAO-A, DYRK1A and cytotoxicity to several select cancer cell lines. Compounds 3e and 4c exhibited an IC50 of 0.83±0.03 and 0.43±0.002μM against MAO-A and an IC50 of 0.26±0.04 and 0.36±0.001μM against MAO-B, respectively. Molecular docking studies revealed π-π interactions between the synthesized molecules and aromatic amino acid residues. Conclusion & future perspective: The current study delineates the structural requirements for MAO-A selectivity and such information may be helpful in designing selective analogs for kinase, DYRK1A and harmine-based cytotoxics without apparent MAO enzyme inhibition.

Characterizing the binding interactions of PFOA and PFOS with catalase at the molecular level

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have effects on human health by inducing oxidative stress and catalase (CAT) is a vital enzyme involved in protection against oxidative damage. The interactions of PFOA and PFOS with CAT were investigated by using biophysical methods including spectroscopic techniques, molecular docking and enzyme activity measurements. UV–visible, circular dichroism (CD) and resonance light scattering (RLS) spectroscopy results showed that the structure and conformation of CAT were changed by PFOA and PFOS. PFOA could loosen and unfold the skeleton of CAT but PFOS affected the microenvironment around the aromatic amino acid residues and heme groups. Both PFOA and PFOS altered the secondary structure of CAT by decreasing α-helix and increasing β-sheet content. The size of CAT was smaller and CAT became dispersed when it was bound by perfluorinated compounds (PFCs). Furthermore, enzyme activity test showed that PFOS decreased the activity of CAT because the binding site of PFOS was close to the active center of CAT, but PFOA had little effect on the activity because PFOA bound at the surface of the enzyme. These results indicated that PFCs could damage the structures and conformations of CAT but the changes were not always related to the activity and function of CAT.

Studies on the interaction of the aromatic amino acids tryptophan, tyrosine and phenylalanine as well as tryptophan-containing dipeptides with cyclodextrins

Hydrolysates of plant or whey proteins, respectively, contain a variety of bioactive peptides which are promising ingredients of functional foods. The application of hydrolysates in foods, however, is limited due to their notable bitter taste, which is attributed to aromatic and hydrophobic amino acid residues. In the present study, NMR and UV-/fluorescence measurements were performed to investigate interactions between cyclodextrins and individual peptides originating from enzymatically hydrolyzed food proteins, namely isoleucyl-tryptophan and tryptophyl-leucine. It was found that the hydrophobic side chain of aromatic amino acids penetrates directly into the cavity of the cyclodextrins. For tryptophan, tyrosine and phenylalanine, the calculated association constants were less than 600 M−1 (1H-NMR), indicating weak–moderate interactions. Tryptophan and β-cyclodextrin formed the most stable complexes. The strength of these complexes increased for tryptophan within the sequence of dipeptides, especially in C-terminal position. Initial investigations on the taste of rice protein hydrolysate have shown that the addition of cyclodextrins except of α-cyclodextrin significantly reduced its bitterness. Especially, the use of β-cyclodextrin was particularly effective. For the first time, we could determine the bitter taste-lowering potential of γ-cyclodextrin, however, less pronounced compared to β-cyclodextrin. Therefore, β-cyclodextrin is the most suitable additive for improving the sensory quality of hydrolysate-containing functional foods.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H–TyrMetTyr–OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

Investigation of the interaction between benzaldehyde thiosemicarbazone compounds and xanthine oxidase

A series of substituted benzaldehyde thiosemicarbazide compounds (1–7) were synthesized as xanthine oxidase (XO) inhibitors, and the interactions between substituted benzaldehyde thiosemicarbazide compounds (1–7) and XO were studied by ultraviolet spectroscopy, fluorescence spectroscopy, and molecular docking. It was found that the hydrogen bond and hydrophobicity were the main interactions between substituted benzaldehyde thiosemicarbazide compounds and XO, and introducing OH at the para position of the benzene ring and a Ph- or Me-group at the amino terminal of compound 4 increased the modifier's inhibitory activity. The results suggest that the newly introduced benzene ring interacted with the hydrophobic cavity of XO by means of the π-π stacking force between the newly introduced benzene ring and the aromatic amino acid residues, such as the Phe residue, which greatly increased the modifier's inhibitory activity. We conclude that introducing the Ph-group at the amino terminal of compound 4 and the OH group at the para position of the benzene ring was a good route to obtain novel XO inhibitors. Fluorescence spectroscopy assisted by 8-anilino-1-naphthalenesulfonic acid fluorescence probing and molecular docking were helpful for achieving a preliminary and relatively clear understanding of the interactions between target compounds and XO, which deserve further study.

Peptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteins fermented using Bacillus subtilis

In this study, tomato seeds were obtained as by-products and submitted to fermentation with the proteolytic strain Bacillus subtilis A14h. The resulting peptide mixture was fractionated and purified through different chromatographic steps. Fractions were assayed for antioxidant and angiotensin converting enzyme (ACE)-inhibitory activities and peptides were identified by using nano-liquid chromatography coupled to mass spectrometry in tandem (nLC-MS/MS). Most of the identified peptides were smaller than 1000 Da and had different aromatic and hydrophobic amino acid residues. Their sequences were novel but some of them showed active domains previously reported in other bioactive peptides. The hexapeptide DGVVYY showed an IC50 value of 2 µM in angiotensin-I converting enzyme (ACE-I) inhibitory activity, whereas the pentapeptide GQVPP displayed a 97% of DPPH activity at 0.4 mM. The results revealed that B. subtilis fermentation of tomato by-products could be a good strategy for obtaining added-value peptides that might be used as an ingredient in functional foods and nutraceuticals.

Peptide Self-Assembly Measured Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a flexible and powerful technique to measure the diffusion of fluorescently labeled particles. It has been important in examining a range of biological processes, from intracellular transport, to DNA hybridization. It is particularly suited to measuring the assembly of peptides, since peptides are often too small to be detected by standard light scattering methods, or may not contain aromatic amino acid residues, which limits the use of other spectroscopic techniques. In this protocol, we describe state-of-the-art sample preparation for Aβ1-42 peptide solutions and the measurement and analysis of the self-assembly of the peptide to form fibrils via a number of intermediate states using FCS.

Molecular Signatures of Cholesterol Interaction with Serotonin Receptors.

The interaction of G protein-coupled receptors (GPCRs) with cholesterol is a hallmark of their function, organization, and structural dynamics. Several cholesterol interaction sites, such as the cholesterol recognition amino acid consensus (CRAC) and cholesterol consensus motif (CCM), have been mapped from crystallography, bioinformatics, and simulation studies. In this article, we characterize common descriptors for cholesterol interaction sites in the serotonin1A receptor from a series of coarse-grain simulations. We have identified a novel interaction mode for cholesterol in which the cholesterol polar headgroup interacts with aromatic amino acid residues, such as tryptophan and tyrosine. The cholesterol rings interact with both aromatic residues and nonpolar residues, thereby constituting a signature aromatic interaction site. In addition, we report a similar binding mode in the crystal structures of the serotonin2B receptor, suggesting that this binding mode could be a general feature of the serotonin receptor family. Interestingly, this signature aromatic interaction site is present along with one of the CRAC motifs in the serotonin1A receptor. Our results represent an important step toward mapping out the diversity of cholesterol-GPCR interaction sites.

Denaturation of Lysozyme with Visible-light-responsive Photocatalysts of Ground Rhodium-doped and Ground Rhodium-antimony-co-doped Strontium Titanate.

Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO3 (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO3 (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.

Aromatic interactions at the ligand-protein interface: Implications for the development of docking scoring functions.

The ability to design and fine-tune non-covalent interactions between organic ligands and proteins is indispensable to rational drug development. Aromatic stacking has long been recognized as one of the key constituents of ligand-protein interfaces. In this communication, we employ a two-parameter geometric model to conduct a large-scale statistical analysis of aromatic contacts in the experimental and computer-generated structures of ligand-protein complexes, considering various combinations of aromatic amino acid residues and ligand rings. The geometry of interfacial π-π stacking in crystal structures accords with experimental and theoretical data collected for simple systems, such as the benzene dimer. Many contemporary ligand docking programs implicitly treat aromatic stacking with van der Waals and Coulombic potentials. Although this approach generally provides a sufficient specificity to model aromatic interactions, the geometry of π-π contacts in high-scoring docking conformations could still be improved. The comprehensive analysis of aromatic geometries at ligand-protein interfaces lies the foundation for the development of type-specific statistical potentials to more accurately describe aromatic interactions in molecular docking. A Perl script to detect and calculate the geometric parameters of aromatic interactions in ligand-protein complexes is available at https://github.com/michal-brylinski/earomatic. The dataset comprising experimental complex structures and computer-generated models is available at https://osf.io/rztha/.

Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris.

The thermophilic bacterium Thermoactinomyces vulgaris strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland, was shown to contain thermoactinoamide A (1), a new cyclic hexapeptide composed of mixed d and l amino acids, along with five minor analogues (2-6). The structure of 1 was determined by one- and two-dimensional NMR spectroscopy, high-resolution tandem mass spectrometry, and advanced Marfey's analysis of 1 and of the products of its partial hydrolysis. Thermoactinoamide A inhibited the growth of Staphylococcus aureus ATCC 6538 with an MIC value of 35 μM. On the basis of literature data and this work, cyclic hexapeptides with mixed d/l configurations, one aromatic amino acid residue, and a prevalence of lipophilic residues can be seen as a starting point to define a new, easily accessible scaffold in the search for new antibiotic agents.

Protease cleavage site fingerprinting by label-free in-gel degradomics reveals pH-dependent specificity switch of legumain.

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.

Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond

BackgroundThe eighth step of l-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in l-histidine biosynthesis.ResultsDeletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete l-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases.Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases.ConclusionThe kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes.