Genome Analysis of a Potential Novel Vibrio Species Secreting pH- and Thermo-Stable Alginate Lyase and Its Application in Producing Alginate Oligosaccharides.

Alginate lyase is an attractive biocatalyst that can specifically degrade alginate to produce oligosaccharides, showing great potential for industrial and medicinal applications. Herein, an alginate-degrading strain HB236076 was isolated from Sargassum sp. in Qionghai, Hainan, China. The low 16S rRNA gene sequence identity (<98.4%), ANI value (<71.9%), and dDDH value (<23.9%) clearly indicated that the isolate represented a potential novel species of the genus Vibrio. The genome contained two chromosomes with lengths of 3,007,948 bp and 874,895 bp, respectively, totaling 3,882,843 bp with a G+C content of 46.5%. Among 3482 genes, 3332 protein-coding genes, 116 tRNA, and 34 rRNA sequences were predicted. Analysis of the amino acid sequences showed that the strain encoded 73 carbohydrate-active enzymes (CAZymes), predicting seven PL7 (Alg1-7) and two PL17 family (Alg8, 9) alginate lyases. The extracellular alginate lyase from strain HB236076 showed the maximum activity at 50 °C and pH 7.0, with over 90% activity measured in the range of 30-60 °C and pH 6.0-10.0, exhibiting a wide range of temperature and pH activities. The enzyme also remained at more than 90% of the original activity at a wide pH range (3.0-9.0) and temperature below 50 °C for more than 2 h, demonstrating significant thermal and pH stabilities. Fe2+ had a good promoting effect on the alginate lyase activity at 10 mM, increasing by 3.5 times. Thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) analyses suggested that alginate lyase in fermentation broth could catalyze sodium alginate to produce disaccharides and trisaccharides, which showed antimicrobial activity against Shigella dysenteriae, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. This research provided extended insights into the production mechanism of alginate lyase from Vibrio sp. HB236076, which was beneficial for further application in the preparation of pH-stable and thermo-stable alginate lyase and alginate oligosaccharides.

Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.

Cloning, expression and characterization of novel hyaluronan lyases Vhylzx1 and Vhylzx2 from Vibrio sp. ZG1.

Hyaluronidases are a class of enzymes that can degrade hyaluronic acid and have a wide range of applications in the medical field. In this study, the marine bacterium Vibrio sp. ZG1, which can degrade HA, was isolated, leading to the discovery of two novel hyaluronan lyases, Vhylzx1 and Vhylzx2, through genome sequencing and bioinformatic analysis. These lyases belong to the polysaccharide lyase-8 family. Vhylzx1 and Vhylzx2 specifically degrade HA, with highest activity at 35 °C, pH 5.7 and 50 °C, pH 7.1. Vhylzx1 and Vhylzx2 are endo-type enzymes that can fully degrade HA into unsaturated disaccharides. Sequence homology assessment and site-directed mutagenesis revealed that the catalytic residues of Vhylzx1 are Asn231, His281, and Tyr290, and that the catalytic residues of Vhylzx2 are Asn227, His277, and Tyr286. Moreover, this study used consensus sequences to enhance the specific activity of Vhylzx2 mutants. Notably, the mutants V564I, N742D, L619F, and D658G increases the specific activity by 2.4, 2.2, 1.3, and 1.2-fold. These characteristics are useful for further basic research and applications, and have a promising application in the preparation of biologically active hyaluronic acid oligosaccharides.

Characterization and structural identification of a novel alginate-specific carbohydrate-binding module (CBM): The founding member of a new CBM family.

Alginate is a commercially important polysaccharide widely distributed in brown algae. Carbohydrate-binding modules (CBMs), a class of commonly used polysaccharide-binding proteins, have greatly facilitated the investigations of polysaccharides. Few alginate-binding CBMs have been hitherto reported and structurally characterized. Herein, an unknown domain from a potential PL6 family alginate lyase in the marine bacterium Vibrio breoganii was discovered and recombinantly expressed. The obtained protein, designated VbCBM106, displayed the favorable specificity to alginate. The unique sequence and well-defined function of VbCBM106 reveal a new CBM family (CBM106). Moreover, the structure of VbCBM106 was determined at a 1.5 Å resolution by the X-ray crystallography, which shows a typical ß-sandwich fold comprised of two antiparallel ß-sheets. Site-directed mutagenesis assays confirmed that positively charged polar residues are crucial for the ligand binding of VbCBM106. The discovery of VbCBM106 enriches the toolbox of alginate-binding proteins, and the elucidation of critical residues would guide the future practical applications of VbCBM106.

A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

A "Pro-Asp-Thr" Amino Acid Repeat from Vibrio sp. QY108 Alginate Lyase Exhibits Alginate-Binding Capacity and Enhanced Soluble Expression and Thermostability.

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.

High-Level Extracellular Production of a Trisaccharide-Producing Alginate Lyase AlyC7 in Escherichia coli and Its Agricultural Application.

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 µg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.

Plasma-activated water improves the accessibility of chitinase to chitin by decreasing molecular weight and breaking crystal structure.

Chitooligosaccharides, the hydrolysis products of chitin, have superior biological activities and application value to those of chitin itself; however, the ordered and highly crystalline structure of chitin renders its degradation by chitinase difficult. Herein, the effects of plasma-activated water (PAW) pre-treatment on the physicochemical properties, crystal structure, and enzymatic hydrolysis of chitin were investigated. The hydrolysis of PAW-pre-treated chitin (PAW activation time of 5 min) using chitinase from Vibrio harveyi (VhChit2) yielded 71 % more reducing sugar, compared with that from untreated chitin, with the degree of chitin hydrolysis increasing from 13 % without pre-treatment to 23 % post-treatment. Moreover, the amount of VhChit2 adsorbed by chitin increased from 41.7 to 58.2 mg/g. Fourier transform infrared spectrometry revealed that PAW could break the ß-1,4-glycosidic bonds of chitin (but had no effects on the hydrogen and amido bonds), thereby decreasing the molecular weight and crystallinity of the polysaccharide, which caused its structural damage and enhanced its enzymatic hydrolysis by chitinase. Consequently, PAW pre-treatment can be considered a simple, effective, and environmentally-friendly method for the biotransformation of chitin as its easier hydrolysis yields high-value products.

Deciphering the Nitrogen Fixation Gene Cluster in Vibrio natriegens: A Study on Optimized Expression and Application of Nitrogenase.

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.

Investigating the Potential of Phenolic Compounds and Carbohydrates as Acceptor Substrates for Levansucrase-Catalyzed Transfructosylation Reaction.

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

Characterization of a Novel Alginate Lyase with Two Alginate Lyase Domains from the Marine Bacterium Vibrio sp. C42.

Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli. All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.

NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Structure of Vibrio collagenase VhaC provides insight into the mechanism of bacterial collagenolysis.

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

Directed evolution of an amine transaminase for the synthesis of an Apremilast intermediate via kinetic resolution.

Apremilast is an important active pharmaceutical ingredient that relies on a resolution to produce the key chiral amine intermediate. To provide a new catalytic and enzymatic process for Apremilast, we performed the directed evolution of the amine transaminase fromVibriofluvialis. Six rounds of evolution resulted in the VF-8M-E variant with > 400-fold increase specific activity over the wildtype enzyme. A homology model of VF-8M-E was built and a molecular docking study was performed to explain the increase in activity. The purified VF-8M-E was successfully applied to produce the key chiral amine intermediate in enantiopure form and 49% conversion via a kinetic resolution, representing a new enzymatic access towards Apremilast.

Structural basis of chitin utilization by a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii strain ATCC BAA-1116.

Vibrio species play a crucial role in maintaining the carbon and nitrogen balance between the oceans and the land through their ability to employ chitin as a sole source of energy. This study describes the structural basis for the action of the GH20 ß-N-acetylglucosaminidase (VhGlcNAcase) in chitin metabolism by Vibrio campbellii (formerly V. harveyi) strain ATCC BAA-1116. Crystal structures of wild-type VhGlcNAcase in the absence and presence of the sugar ligand, and of the unliganded D437A mutant, were determined. VhGlcNAcase contains three distinct domains: an N-terminal carbohydrate-binding domain linked to a small α+ß domain and a C-terminal (ß/α)8 catalytic domain. The active site of VhGlcNAcase has a narrow, shallow pocket that is suitable for accommodating a small chitooligosaccharide. VhGlcNAcase is a monomeric enzyme of 74 kDa, but its crystal structures show two molecules of enzyme per asymmetric unit, in which Gln16 at the dimeric interface of the first molecule partially blocks the entrance to the active site of the neighboring molecule. The GlcNAc unit observed in subsite -1 makes exclusive hydrogen bonds to the conserved residues Arg274, Tyr530, Asp532 and Glu584, while Trp487, Trp546, Trp582 and Trp505 form a hydrophobic wall around the -1 GlcNAc. The catalytic mutants D437A/N and E438A/Q exhibited a drastic loss of GlcNAcase activity, confirming the catalytic role of the acidic pair (Asp437-Glu438).

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.