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Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2026 Sep 16;82(Pt 10):336–347. doi: 10.1107/S2053230X2600868X

Structural insights into a putative BLF1-like deamidase from Methylomonas sp. Kb3

Nur Afiqah Shalihin Muhamad Ismail a, Amirul Adli Abd Aziz b, Atsushi Nakagawa c, Farah Diba Abu Bakar a, Abdul Munir Abdul Murad a, Sheila Nathan a, Mohd Firdaus Mohd Raih b, Doris Huai Xia Quay d,*, David Rice e
Editor: S Sherifff
PMCID: PMC13629640  PMID: 42745721

The crystal structure of a putative BLF1-like deamidase from Methylomonas sp. Kb3 reveals conservation of the catalytic cysteine–histidine dyad despite extensive sequence variation. These findings expand the diversity of BLF1-like deamidases and provide a structural framework for understanding their evolution and potential functions.

Keywords: BLF1-like deamidases, X-ray crystallography, protein structure, Methylomonas sp. Kb3, cysteine-histidine catalytic dyad

Abstract

Bacteria that act as eukaryotic pathogens are known to express enzyme toxins that interfere with critical pathways in their targets. Among these are deamidases that modify conserved glutamine residues such as those found in eukaryotic initiation factor 4A (eIF4A) or in the small GTPases Rho, Rac and Cdc42, converting them to glutamic acid and thereby disrupting the function of the target proteins. Structural and biochemical studies have shown that deamidases from Burkholderia, Burkholderia lethal factor 1 (BLF1), and from Escherichia coli, cytotoxic necrotizing factor (CNF1), share a similar fold in their deamidase domains with similar mechanisms for deamidase chemistry. The active site of these toxins contains an apparently conserved LSGC motif as part of a cysteine–histidine catalytic dyad which catalyses the site-specific deamidation. Sequence searches in bacterial databases have suggested that deamidases with this fold are more widespread in nature and that the LSGC motif varies substantially. Using crystals grown in magnesium sulfate, we determined the 1.95 Å resolution crystal structure of a putative deamidase from Methylomonas sp. Kb3, CWO84_RS12955, which shows limited sequence identity to BLF1. The structure reveals that CWO84_RS12955 adopts a similar fold to that of BLF1, albeit with significant differences at the periphery due to sequence insertions and deletions, and that it conserves the catalytic dyad but replaces the LSGC motif with MDGC. However, compared with BLF1, none of the residues that interact with eIF4A are conserved. These structural features suggest that the Methylomonas protein that we have identified may act as a BLF1-like deamidase but against a currently unknown target, and that other bacterial proteins with similar sequence fingerprints could play important roles in broader bacterial biology.

1. Introduction

Bacterial deamidases are enzymes that catalyse the site-specific deamidation of glutamine or asparagine residues to glutamic acid and aspartic acid, respectively, thereby altering the function of the target protein (Washington et al., 2013 ▸). The first member of this family to be identified was cytotoxic necrotizing factor 1 (CNF1), a 108 kDa protein toxin produced by pathogenic Escherichia coli. CNF1 permanently activates Rho-family GTPases by deamidating Gln63 in Rho and Gln61 in Rac and Cdc42, converting them to glutamic acid (Boquet, 2001 ▸; Fabbri et al., 2010 ▸; Flatau et al., 1997 ▸). This modification blocks intrinsic GTP hydrolysis and GTPase-activating protein (GAP)-stimulated activity, leading to constitutive activation of these regulators (Boquet, 1998 ▸; Flatau et al., 1997 ▸). CNF1 is organized as an A–B-type toxin with an N-terminal receptor-binding domain, a central translocation domain and a C-terminal catalytic domain (Boquet, 2001 ▸). Functionally, CNF1 causes dramatic changes in the actin cytoskeleton, including the formation of stress fibres and focal adhesions, and it was the first bacterial toxin discovered to act through protein deamidation (Boquet, 2001 ▸; Flatau et al., 1997 ▸). The cytotoxic necrotizing factors (CNFs) represent an expanding family of bacterial deamidating toxins that includes CNF1, CNF2 and CNF3 from E. coli, as well as CNFY from Yersinia pseudotuberculosis (Knust & Schmidt, 2010 ▸; Stoll et al., 2009 ▸). CNFY has been crystallized and confirmed as a glutamine deamidase based on its conserved cysteine and histidine residues, and it shares 61% amino-acid identity with CNF1 distributed across the full protein sequence (Blumenthal et al., 2007 ▸). Despite their structural similarity, CNF1 and CNFY interact with different host receptors and display distinct cell-type specificities, although both exploit the endosomal pathway for cytosolic entry (Blumenthal et al., 2007 ▸).

Subsequent to the characterization of CNF1, a bacterial deamidase, Burkholderia lethal factor 1 (BLF1), was identified as a toxin produced by the pathogen Burkholderia pseudomallei that inhibits eukaryotic protein synthesis by targeting the RNA helicase eIF4A and deamidating Gln339 (Cruz-Migoni et al., 2011 ▸). This modification leads to the abolition of the helicase activity of eIF4A, thereby blocking translation initiation (Cruz-Migoni et al., 2011 ▸; Mobbs et al., 2022 ▸). BLF1 is selectively toxic to MYCN-amplified neuroblastoma cells while sparing nontransformed cells, highlighting its potential as a targeted anticancer therapeutic with limited off-target effects (Rust et al., 2018 ▸). Structural studies showed that BLF1 and the catalytic deamidase domain of CNF1 adopt a similar β-sandwich fold, despite sharing less than 20% sequence identity (Cruz-Migoni et al., 2011 ▸). The structure of a BLF1C94S–eIF4A complex revealed that the toxin binds in the cleft between the RecA-like domains of eIF4A, positioning the target glutamine beside the conserved cysteine–histidine dyad (Mobbs et al., 2022 ▸). In both BLF1 and CNF1 the catalytic cysteine is part of a conserved LSGC motif, but sequence differences flanking the catalytic site residues determine their differential substrate specificity.

Database searches using BLF1 as a query identified numerous putative BLF1-like proteins with varying levels of sequence identity that retain the conserved catalytic cysteine and histidine residues characteristic of the BLF1-like deamidase family. This conservation suggests that BLF1-like proteins belong to an evolutionarily conserved family of deamidases that is widely distributed among bacteria. One such protein was identified in a plant-pathogenic Xanthomonas species (Muhamad Ismail et al., 2023 ▸). Computational modelling of a predicted Xanthomonas protein revealed only 19% sequence identity to BLF1. Despite this low sequence similarity, the protein was predicted to adopt a similar BLF1-like deamidase fold while retaining the conserved LSGC catalytic motif and the nearby catalytic histidine (Muhamad Ismail et al., 2023 ▸). Although these structural predictions support its classification as a BLF1-like deamidase, no experimental structural and biochemical characterization has been reported, and its biological function remains unknown. The preservation of catalytic motifs despite extensive sequence divergence is consistent with observations in other cysteine-dependent deamidase families, where catalytic residues are highly conserved while surrounding regions evolve to accommodate differences in substrate recognition and biological function (Mobbs et al., 2022 ▸; Washington et al., 2013 ▸).

With the aim of deepening our understanding of the deamidase family, in this paper we report the structural characterization of CWO84_RS12955 from the gene encoding a putative BLF1-like deamidase from Methylomonas sp. Kb3. Although CWO84_RS12955 shares less than 10% sequence identity with BLF1, it retains key sequence features associated with the BLF1 family, including a putative catalytic motif in which the canonical LSGC sequence found in BLF1 and CNF1 is replaced by an MDGC motif. The conservation of key catalytic residues despite extensive sequence divergence suggests a possible evolutionary relationship between CWO84_RS12955 and established bacterial deamidases.

Methylomonas sp. Kb3 is a nonpathogenic aerobic methanotroph isolated from rice-field rhizospheric soil in western India and belongs to a genus commonly found in aquatic habitats, wetlands and rice-field ecosystems (Rahalkar & Pandit, 2018 ▸). Given the ecological importance of methanotrophs in global carbon cycling and their distribution across diverse and dynamic environments, these bacteria experience a range of environmental conditions that may shape their physiological and metabolic adaptation (Rahalkar et al., 2021 ▸). As key mediators of methane oxidation in soil and aquatic systems, methanotrophs contribute significantly to carbon flux regulation within microbial ecosystems (Guerrero-Cruz et al., 2021 ▸). Within these complex microbial communities, methanotrophs coexist and interact with diverse microorganisms through both cooperative and competitive relationships, including metabolic cross-feeding, competition for nutrients and responses to metabolites produced by neighbouring microorganisms. Such interactions can influence methanotroph growth, physiology and community structure (Brenzinger et al., 2024 ▸).

The identification of a putative BLF1-like deamidase in a nonpathogenic environmental bacterium is particularly intriguing, given that bacterial deamidases have predominantly been characterized in the context of virulence and host–pathogen interactions (Flatau et al., 1997 ▸; Schmidt et al., 1997 ▸; Washington et al., 2013 ▸). Its presence in Methylomonas sp. Kb3 therefore raises the possibility that members of this enzyme family may perform broader biological functions beyond pathogenicity, potentially contributing to interactions with other organisms or providing a competitive advantage under particular ecological conditions. However, the natural biological target and ecological function of CWO84_RS12955 remain unknown and require further investigation. The structural insights provided by this work establish a framework for investigating the catalytic mechanism of CWO84_RS12955 and for comparative analyses with BLF1. Such comparisons may reveal conserved structural features, identify determinants of substrate specificity and broaden our understanding of the structural and evolutionary diversity of BLF1-like deamidases.

2. Materials and methods

2.1. Identification of a putative BLF1-like deamidase from Methylomonas sp. Kb3 by sequence analysis

To identify proteins distantly related to BLF1 and expand the diversity of homologs beyond closely related sequences, the amino-acid sequence of BLF1 from B. pseudomallei was used as a query in Position-Specific Iterated BLAST (PSI-BLAST) searches against the NCBI nonredundant (nr) protein database. The search was initiated using either the entire or a partial BLF1 protein sequence. Hits were inspected manually and shortlisted based on E-value, percentage identity, query coverage and the presence of conserved amino-acid residues potentially associated with BLF1-like deamidase activity. Protein candidates were further assessed by sequence alignment against BLF1 to determine whether they retained residues previously implicated in catalysis and substrate recognition. In particular, emphasis was placed on residues corresponding to the catalytic cysteine and histidine reported to contribute to BLF1-like glutamine deamidation of eIF4A. Among the proteins identified, one candidate from Methylomonas sp. Kb3 was selected for further study. The selected candidate was designated as a putative BLF1-like deamidase and was subjected to subsequent structural and comparative analyses.

2.2. Macromolecule production

The CWO84_RS12955 gene from Methylomonas sp. Kb3 was synthesized and cloned into the His6 LIC cloning vector 2B-T to produce a recombinant protein with a TEV-cleavable N-terminal 6×His tag (Table 1 ▸). The vector (2B-T) was a gift from Scott Gradia (Addgene plasmid #29666; https://n2t.net/addgene/29666; RRID Addgene_29666). The recombinant plasmid was transformed into E. coli strain BL21 (DE3) Star cells. A single transformant colony was inoculated into Luria–Bertani (LB) autoinduction medium supplemented with 100 µg ml−1 ampicillin and incubated at 37°C with shaking at 250 rev min−1 for 4 h. The culture was then shifted to 25°C and incubated overnight to facilitate protein expression before harvesting the cells. The bacterial pellet was resuspended in a lysis buffer consisting of 50 mM Tris–HCl pH 7.5, 500 mM sodium chloride, 30 mM imidazole, 20 mM β-mercapto­ethanol. Cell disruption was performed using a sonicator (JY96-IIN) with 40 cycles at 40% amplitude, each lasting 30 s. The soluble fraction was separated from cell debris by centrifugation at 25 155g and 4°C for 1 h. The supernatant was filtered through a 0.4 µm PVDF membrane filter and loaded onto a 5 ml HisTrap HP column (Ni2+–NTA affinity column, GE Healthcare, USA) pre-equilibrated with the lysis buffer. The binding buffer used was identical to the lysis buffer.

Table 1. Macromolecule-production information for the putative BLF1-like deamidase CWO84_RS12955.

Source organism Methylomonas sp. Kb3
DNA source Chemically synthesized (IDT gene, Apical)
Expression vector 2B-T
Plasmid construction method Ligation-independent cloning (LIC)
Forward primer 5′-TACTTCCAATCCAATGCAATGACCGCAATTTCTGATTTTG-3′
Reverse primer 5′-TTATCCACTTCCAATGTTATTATTAGACATTTTTTAGCGC-3′
Expression host E. coli strain BL21 (DE3) Star
Complete amino-acid sequence of the construct produced† mksshhhhhhenlyfq/snaMTAISDFVANPKQFLKNNVLRVLFTMPAQPNSIGMFKFERKNYDAVKLATGANIPCYSLVPIHGQEATIFSRNATSANRDYLRAYWCPYEDDAMHSIMVGAGADFMFTSNMDGCSFGVGSATPTGDRRVAHINLRSQPNSHNLQDGTLAVQSLTDHHVKPDRYMKSSRTPGSIPGEIKATTIGIRNTATGAWSFHYQQYRLLGGQINQVYLLALKNV
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†

Lower case letters indicate the vector sequence consisting of an N-terminal 6×His affinity tag and TEV protease recognition sequence introduced for recombinant expression. The slash (/) denotes the TEV protease cleavage site (ENLYFQ↓S). The affinity tag was removed by TEV protease digestion prior to crystallization, leaving three vector-derived residues (sna) at the N-terminus of the recombinant protein.

The protein was eluted using a linear gradient to 50 mM Tris–HCl pH 7.5, 500 mM sodium chloride, 500 mM imidazole, 20 mM β-mercaptoethanol. The target protein eluted at approximately 500 mM imidazole. Around 25 ml of fractions containing the protein were pooled and incubated with TEV protease at a ratio of 1:3 (TEV protease:protein, molar ratio). The pooled fractions were placed into a dialysis tube overnight using a 3.5 kDa molecular-weight cutoff (SnakeSkin) bag in 2 l dialysis buffer consisting of 50 mM Tris–HCl pH 7.5, 500 mM sodium chloride, 20 mM β-mercaptoethanol. The following day, the cleaved protein mixture underwent a second IMAC purification step to separate the target protein from the His-tag and TEV protease.

The protein was collected in the flowthrough and wash fractions using binding buffer. These fractions were pooled and concentrated with a Vivaspin concentrator fitted with a 3 kDa molecular-weight cutoff filter (Sartorius, Germany). The concentrated protein was further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 pg column (GE Healthcare, USA) pre-equilibrated in 50 mM Tris–HCl pH 7.5, 100 mM sodium chloride, 20 mM β-mercaptoethanol. Protein purity and apparent molecular weight were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), which showed an apparent molecular weight of approximately 24 kDa consistent with the theoretical molecular weight (Supplementary Fig. S1). Coomassie Brilliant Blue R-250 was used for staining, and the protein concentration was determined using a NanoDrop spectrophotometer based on UV–Vis absorbance.

2.3. Crystallization

The purified CWO84_RS12955 was concentrated to 12 mg ml−1 using a Vivaspin concentrator equipped with a 3 kDa molecular-weight cutoff filter (Sartorius, Germany). Initial crystallization trials were conducted using the sitting-drop vapour-diffusion method in 96-well plates (Molecular Dimensions), employing the Crystal Screen and Crystal Screen 2 kits (Hampton Research). Initial crystals were observed after three days of incubation at 4°C in a condition consisting of 1.6 M ammonium sulfate, 0.1 M MES monohydrate pH 6.5, 10%(v/v) 1,4-dioxane. A second crystallization condition, consisting of 0.1 M MES monohydrate pH 6.5, 1.6 M magnesium sulfate, produced crystals after seven days. Both conditions were selected for optimization using the hanging-drop vapour-diffusion method with a protein:reservoir solution ratio of 2:1. The second crystallization condition consistently produced well formed crystals suitable for X-ray diffraction data collection at SPring-8, Japan (Supplementary Fig. S2). Under the optimized conditions, crystals reached a maximum size of approximately 200 µm within two weeks. Details of the crystallization conditions are summarized in Table 2 ▸.

Table 2. Crystallization of CWO84_RS12955.

Method Sitting-drop vapour diffusion for initial crystal screening and hanging-drop vapour diffusion for crystal optimization
Plate type 96-well plates for initial crystal screening and 24-well plates (Hampton Research HR3-107) for crystal optimization
Temperature (°C) 4
Protein concentration (mg ml−1) 12
Buffer composition of protein solution 50 mM Tris–HCl pH 7.5, 100 mM sodium chloride, 20 mM β-mercaptoethanol
Composition of reservoir solution 0.1 M MES monohydrate pH 6.5, 1.6 M magnesium sulfate heptahydrate
Volume and ratio of drop 1.0 µl for initial crystal screening and 2.0 µl for optimization; 1:1 and 2:1 protein:reservoir solution
Volume of reservoir 50 µl for initial crystal screening and 1.0 ml for optimization
Composition of the cryoprotectant 0.1 M MES monohydrate pH 6.5, 1.6 M magnesium sulfate heptahydrate, 30% ethylene glycol
Drop setting Drop setting was performed manually using a micropipette. Crystallization drops were prepared by mixing protein and reservoir solutions at the specified ratios of 1:1 (screening) and 2:1 (optimization).
Seeding No
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2.4. Data collection and processing

Crystals of CWO84_RS12955 were soaked in cryoprotectant for 5 min, flash-cooled in liquid nitrogen and used for X-ray diffraction data collection (Table 2 ▸). The data were processed and indexed using XDS (Kabsch, 2010 ▸) and were scaled and merged using AIMLESS (version 0.8.2; Evans & Murshudov, 2013 ▸). The merged intensities were converted to structure-factor amplitudes using CTRUNCATE (version 1.17.29; Winn et al., 2011 ▸) with French–Wilson treatment prior to molecular replacement and refinement. Data-collection and processing statistics are summarized in Table 3 ▸.

Table 3. Data-collection and processing statistics for CWO84_RS12955.

Values in parentheses are for the outer shell.

Diffraction source SPring-8, Hyogo, Japan
Wavelength (Å) 1.000
Temperature (K) 100
Detector EIGER X 16M
Crystal-to-detector distance (mm) 225
Rotation range per image (°) 0.1
Total rotation range (°) 200
Exposure time per image (s) 0.05
Space group P4132
a, b, c (Å) 168.7, 168.7, 168.7
α, β, γ (°) 90, 90, 90
Mosaicity (°) 0.07
Resolution range (Å) 46.77–1.95 (2.00–1.95)
Total No. of reflections measured 2961873 (176189)
No. of unique reflections 60051 (4178)
Completeness (%) 100 (100)
Multiplicity 44.8 (42.2)
Mean I/σ(I) 24.5 (2.1)
CC1/2 1.0 (0.715)
R merge 0.122 (3.504)
R meas 0.124 (3.546)
R p.i.m. 0.018 (0.542)
Overall B factor from Wilson plot (Å2) 37
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2.5. Protein structure determination

Protein structure determination was carried out using the CCP4 suite (version 9.0.006; Agirre et al., 2023 ▸) and CCP4 Cloud (version 1.8.005; Krissinel et al., 2022 ▸). The structure of CWO84_RS12955 was solved by molecular replacement with Phaser (version 2.8.3; McCoy, 2007 ▸) using an AlphaFold-predicted model of CWO84_RS12955 as the search template. The AlphaFold model was generated using the AlphaFold server powered by AlphaFold3 (Abramson et al., 2024 ▸; https://alphafoldserver.com) from the amino-acid sequence encoded by the CWO84_RS12955 gene (accession No. WP_101053078.1), retrieved from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). Prior to molecular replacement, the AlphaFold model was processed using the MOLREP model-preparation protocol (Vagin & Teplyakov, 1997 ▸) implemented in CCP4 Cloud to generate an optimized search model. Molecular-replacement analysis identified the correct space group as P4132 rather than its enantiomorph P4332. Matthews coefficient analysis indicated the presence of a single molecule in the asymmetric unit. The crystal exhibited an unusually high solvent content of 85.2%, which is considerably higher than that typically observed for protein crystals. Despite this high solvent content, the crystal diffracted to 1.95 Å resolution, enabling accurate model building and refinement.

Initial model building was performed with ModelCraft (version 5.0.2; Bond & Cowtan, 2022 ▸), followed by iterative cycles of refinement using REFMAC5 (version 5.8.0430; Murshudov et al., 2011 ▸). Refinement included anisotropic atomic displacement parameter (B-factor) refinement together with translation–libration–screw (TLS) parameterization using automatically defined TLS groups. The final deposited model contains full atomic B factors, including TLS contributions. H atoms were included in calculated positions using the riding-hydrogen model implemented in REFMAC5 during refinement. Manual model building and the addition of water molecules and ligands based on the electron-density maps were carried out in Coot (version 0.9.8.95; Emsley et al., 2010 ▸). Structural figures were prepared using CCP4MG (version 2.10.11; McNicholas et al., 2011 ▸). The final model was assessed using the wwPDB validation server (https://validate-rcsb-1.wwpdb.org/), including evaluation of the Ramachandran plot, clashscore, rotamer outliers and MolProbity score.

Regions lacking interpretable electron density were not included in the final model. Several prominent positive residual features remained in the final (mFo − DFc) map. The strongest peak, at approximately 40 r.m.s.d., was located on a crystallographic threefold axis near Pro138 and Asn139. Its unusually large apparent height may be enhanced by the superposition of symmetry-related residual density at the crystallographic threefold symmetry element. For residual peaks above 10 r.m.s.d., sulfate, MES, β-mercaptoethanol and chloride were evaluated as possible sources of the density. However, none of these components could be modelled satisfactorily based on the shape of the density and the local chemical environment.

An anomalous difference map was calculated using separately retained Friedel pairs. Peaks were observed at 3 r.m.s.d. at several methionine and cysteine S atoms and at the S atoms of the three modelled sulfate ions, demonstrating that anomalous density was detectable at some ordered sulfur-containing sites. The anomalous map was then inspected at the strongest residual peak and at the other prominent positive (mFo − DFc) peaks down to approximately 7 r.m.s.d.. These unmodelled sites showed either no corresponding anomalous density or only small, fragmented features at 3 r.m.s.d. that disappeared at higher contour levels. Thus, the anomalous map did not support assignment of the residual features to sulfate or another specific anomalous scatterer. Together with the poor agreement between the residual density and the shapes and local chemical environments expected for the tested crystallization and buffer components, these findings did not support the assignment of the features to any of those components. Their identities therefore remain uncertain and the sites were left unmodelled in the final structure. The refined atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 9vfk. The final refinement statistics, including Rwork, Rfree and geometric parameters, are summarized in Table 4 ▸.

Table 4. Refinement statistics for CWO84_RS12955.

Values in parentheses are for the outer shell.

Resolution range (Å) 46.77–1.95 (2.00–1.95)
Completeness (%) 100 (100)
No. of reflections, working set 59975 (4135)
No. of reflections, test set 3019 (232)
Final Rcryst 0.160 (0.340)
Final Rfree 0.176 (0.380)
No. of non-H atoms
 Macromolecules 1705
 Ligands 8 EDO, 15 sulfate
 Waters 189
 Total 1917
R.m.s. deviations from ideality
 Bond lengths (Å) 0.013
 Angles (°) 2.086
Average B factors (Å2)
 Overall 45
 Macromolecules 44
 Ligands EDO, 47; sulfate, 57
 Waters 54
Ramachandran plot
 Favoured regions (%) 97.7
 Outliers (%) 0.0
PDB code 9vfk
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2.6. Structure analysis

Structural visualization and inspection of the overall fold, secondary-structure elements and active-site architecture of CWO84_RS12955 were performed using CCP4MG (version 2.10.11; McNicholas et al., 2011 ▸). Structural comparisons with previously reported deamidases were carried out using TM-align (Zhang & Skolnick, 2005 ▸) to assess fold similarity. TM-align was used to generate structural superimpositions and calculate quantitative measures of structural similarity, including the TM-score, root-mean-square deviation (r.m.s.d.) and number of aligned residues. Sequence alignments and conservation of catalytic residues were analysed using MEGA12 (Kumar et al., 2024 ▸) and ESPript 3.0 (Robert & Gouet, 2014 ▸).

Structural superimpositions between CWO84_RS12955 and BLF1 were performed using UCSF ChimeraX (version 1.9; Pettersen et al., 2021 ▸).

The superimposed structures were analysed to assess the conservation of the overall fold, identify insertion–deletion regions and evaluate the conservation of putative catalytic residues. To evaluate potential substrate recognition and interaction geometry, CWO84_RS12955 was superimposed onto the structure of the BLF1C94S–eIF4A complex (PDB entry 7ppz; Mobbs et al., 2022 ▸). Interface contacts and intermolecular interactions were further inspected using CCP4MG (version 2.10.11; McNicholas et al., 2011 ▸) and PDBePISA (Krissinel & Henrick, 2007 ▸).

3. Results

3.1. Sequence search for BLF1-like deamidases in bacterial databases

The sequence search revealed a significant number of proteins containing low-level similarities to BLF1 across a wide range of bacterial species, particularly belonging to the alpha-, beta- and gammaproteobacterial lineages (Fig. 1 ▸). The sequence identity was most significant close to the region containing the active-site cysteine and histidine residues, although a breakdown occurs of the previously noted sequence motif, LSGC, containing the active-site cysteine. The LSGC motif appeared to be replaced by a number of variants, including MDGC, TNGC and FSNC. Amongst the retrieved hits with an MDGC motif, a sequence from Methylomonassp. Kb3 (CWO84_RS12955-encoding gene) sharing less than 10% sequence identity with BLF1 was identified, indicating substantial sequence divergence (Fig. 1 ▸). Despite this low sequence identity, residues corresponding to the catalytic cysteine–histidine dyad were conserved, suggesting that CWO84_RS12955 may belong to the BLF1-like deamidase family. To further investigate this possibility, a structural study was initiated to assess the conservation of catalytic residues and structural features.

Figure 1.

Figure 1

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Sequence alignment of B. pseudomallei BLF1 and E. coli CNF1 with putative deamidases from other bacteria, focusing on the most highly conserved region. Identical residues are highlighted in red boxes, and conservative substitutions as defined by ESPript3 are shown as red letters outlined in blue. The catalytic cysteine–histidine dyad can be seen to be conserved, with Cys94 and His106 in BLF1 corresponding to Cys114 and His131 in CWO84_RS12955 from Methylomonas sp. Kb3.

3.2. Overall structure of CWO84_RS12955

The final structural model contains CWO84_RS12955 from residue 1 to residue 217 and only a single N-terminal vector-derived alanine residue, as no interpretable electron density was observed for the remaining non-native residues. The electron-density map was well defined, and model validation showed excellent stereochemistry, with 97.7% of residues falling within favoured Ramachandran regions. In particular, the catalytic MDGC motif (Met111-Asp112-Gly113-Cys114) and His131 were clearly resolved in the 2Fo − Fc electron-density map contoured at 2.0 r.m.s.d, supporting the placement of these residues in the final refined model (Fig. 2 ▸). Cys114 was identified as being oxidized to sulfenic acid. Ligands and solvent molecules were clearly resolved and confidently modelled (Table 4 ▸). Notably, the sulfate ions are bound to a positively charged surface patch. Given the high sulfate concentration used during crystallization, this binding is most likely to be a crystallization artefact. Nevertheless, the sulfate-binding site highlights a region of positive electrostatic potential, although there is currently insufficient evidence to assign a biological function to this surface.

Figure 2.

Figure 2

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Electron density surrounding the catalytic residues of CWO84_RS12955. The 2Fo − Fc electron-density map is shown as a blue mesh contoured at 2.0 r.m.s.d.. (a) Electron density surrounding the catalytic MDGC motif (Met111-Asp112-Gly113-Cys114). (b) Electron density surrounding the catalytic residue His131. The continuous and well defined density supports the placement of the catalytic residues in the refined structure.

3.3. Structural similarity between CWO84_RS12955 and BLF1

The crystal structure of CWO84_RS12955 (PDB entry 9vfk) comprises five α-helices and two β-sheets formed by 14 β-strands (Fig. 3 ▸a). Structural superposition with BLF1 (PDB entry 6rvu) yielded an r.m.s.d. of 3.02 Å over 146 equivalent Cα atoms with a TM-score of 0.62 (Fig. 3 ▸b). Structure-based sequence alignment further supports this observation, revealing conservation of the overall fold despite local variations arising from insertions and deletions (Fig. 3 ▸c). The active-site Cys94 of BLF1, which is part of the LSGC motif, aligns well with Cys114 in CWO84_RS12955 despite the fact that in the structure this residue is present in an oxidized form which is thought to be an artefact that arose during protein isolation. Equally, the catalytic His106 of BLF1 and His131 in CWO84_RS12955 overlap very well, thereby apparently preserving the catalytic dyad in both proteins. Interestingly, whilst the cysteine is part of the LSGC motif in BLF1 (and CNF1), this motif becomes MDGC (residues 111–114) in CWO84_RS12955 but with the sequence changes having little effect on the local conformation of these two regions, which remain very similar (Fig. 4 ▸). Indeed, the similarity between the two structures in this region includes the conservation of a critical tyrosine in BLF1 (Tyr164) with Tyr163 in CWO84_RS12955, both of which form a hydrogen bond to the main-chain carbonyl O atom of the catalytic cysteine residue. This local structural similarity, together with the overall similarity in fold, strongly suggests that CWO84_RS12955 acts as a deamidase.

Figure 3.

Figure 3

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(a) Overall three-dimensional structure of CWO84_RS12955 from Methylomonas sp. Kb3 (PDB entry 9vfk) shown in ribbon representation. Selected side chains are displayed as coral sticks (oxygen, red; nitrogen, blue; sulfur, yellow). Secondary-structure elements are coloured as follows: β-strands and β-bulges, blue; 310-turns, pink; 4-turns, tan; 5-turns, coral; α-helices, red; loops, grey. (b) Structural superposition of CWO84_RS12955 (PDB entry 9vfk; coral) and BLF1 (PDB entry 6rvu; lawn green). The black box highlights the overlapping catalytic active sites (MDGC in CWO84_RS12955 and LSGC in BLF1). The alignment yields an r.m.s.d. of 3.02 Å over 146 aligned Cα atoms. (c) Structure-based sequence alignment between CWO84_RS12955 (PDB entry 9vfk) and BLF1 (PDB entry 6rvu). Secondary-structure elements are shown above and below the aligned sequences: coils represent α-helices and arrows represent β-strands. White letters in red-filled boxes indicate identical residues, while red letters in unfilled boxes represent residues with similar biochemical properties.

Figure 4.

Figure 4

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Superposition of conserved residues in the glutamine deamidase BLF1 (lawn green, PDB entry 6rvu) with the putative BLF1-like deamidase CWO84_RS12955 (coral). (a) The MDGC motif of CWO84_RS12955 is structurally aligned with the canonical LSGC motif in BLF1, indicating their similar fold. (b) Structural superposition of the catalytic region of BLF1 with its equivalent in CWO84_RS12955, highlighting the apparent conservation of the His–Cys pair in the active site and including an important tyrosine that forms hydrogen bonds to the cysteine main-chain carbonyl oxygen. Equivalent residues including Cys94/Cys114, His106/His131, Gly93/Gly113, Tyr163/Tyr164 and neighbouring residues (Leu91, Ser92, Met111, Asp112 and Tyr163) are shown as sticks. Dashed lines indicate potential hydrogen-bonding or stabilizing interactions within the active-site region, illustrating the structural similarity between the two proteins. Residue numbering corresponds to each respective protein sequence. O, N and S atoms are coloured red, blue and yellow, respectively.

Superposition of CWO84_RS12955 with BLF1C94S in its complex with human eIF4A (BLF1C94S–eIF4A; PDB entry 7ppz; Mobbs et al., 2022 ▸) showed that the region surrounding the catalytic site superposes well (Fig. 5 ▸a), with the catalytic residues Cys114 and His131 of CWO84_RS12955 adopting positions similar to those of Cys94 and His106 in BLF1. In the BLF1C94S–eIF4A complex the amide N and O atoms of the carboxamide of Gln339 in eIF4A are involved in hydrogen bonds to the carbonyl of Tyr90 and the π-electron system of Trp66 and to the main-chain NH groups of Ser92 and Cys/Ser94 of the LSGC motif in BLF1, respectively (Mobbs et al., 2022 ▸). In CWO84_RS12955, in addition to the conservation of the residues equivalent to the catalytic dyad and Tyr90, the similar conformation of the MDGC motif main chain (Fig. 4 ▸) would permit an equivalent pattern of hydrogen bonding to that seen in BLF1 (Fig. 5 ▸b). Furthermore, Trp66 of BLF1 is replaced by a similarly positioned tyrosine residue (Tyr89) in CWO84_RS12955, allowing its π-electron system to fulfil a similar role. These observations suggest that the active site of CWO84_RS12955 is structurally arranged to interact with a substrate amide, indicating preservation of the active-site geometry required for deamidation.

Figure 5.

Figure 5

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Superposition of the structure of CWO84_RS12955 and that of BLF1C94S in complex with eIF4A (PDB entry 7ppz). (a, b) Superposition of CWO84_RS12955 (shown in coral) with BLF1C94S (shown in lawn green) in complex with human eIF4A (shown in gold), displayed as ribbon representations. The red boxed region highlights the similar orientation of the active site in the two proteins. The catalytic residues Cys114 and His131 of CW084_RS12955 and Cys94 and His106 of BLF1 are shown relative to Gln339 of eIF4A. Equivalent residues from the two structures superpose closely, including Thr108/Thr88 and Tyr163/Tyr164, highlighting the conservation of the interaction geometry. In addition, Tyr89 of CWO84_RS12955 aligns with Trp66 of BLF1, suggesting a conserved positioning of an aromatic residue that may contribute to substrate stabilization through π-mediated interactions. Hydrogen bonds and polar contacts are indicated by dashed lines with distances in Å. C, O, N and S atoms are coloured green, red, blue and yellow, respectively.

3.4. eIF4A-binding residues in BLF1 are not conserved in CWO84_RS12955

BLF1 binds within the cleft formed between the two RecA-like domains of eIF4A, establishing interactions with residues from both domains as well as with the scissile amide bond of the target residue, Gln339, located adjacent to the toxin’s active site (Mobbs et al., 2022 ▸). Formation of the BLF1C94S–eIF4A complex involves two distinct patches on the surface of BLF1 that interact with opposing faces of the N- and C-terminal RecA domains of eIF4A across the wide interdomain groove of the helicase (Mobbs et al., 2022 ▸). These interactions are primarily mediated by an extensive network of complementary polar contacts. In particular, several negatively charged residues in BLF1 (Glu22, Glu70, Asp108, Glu124, Glu127 and Asp128) form electrostatic interactions with positively charged residues in eIF4A (Arg110, Arg161, Arg316, Arg362 and Arg365) (Mobbs et al., 2022 ▸). However, structure-based sequence alignment of CWO84_RS12955 with BLF1 reveals little to no conservation of these key negatively charged residues, suggesting that CWO84_RS12955 may lack the same eIF4A-binding capability or employ a different interaction mechanism.

The interface between BLF1 and the C-terminal domain of eIF4A also involves interactions with the loop containing the target Gln339 in eIF4A together with residues flanking it, revealing that their interactions are mediated extensively by side chains (Mobbs et al., 2022 ▸). Those residues critical to the recognition include the side chains of Asp337, Gln339 and Gln340 in eIF4A, which point outwards from its molecular surface, binding into the pocket provided by BLF1, where major contributions arise from the five side chains of Trp66, Tyr89, Arg142, Gln145 and Asn168, together with main-chain interactions from Cys/Ser94, Ser92 and Tyr90. Whilst the structure of CWO84_RS12955 would allow the main-chain interactions to be maintained, none of these five side chains are found in the sequence of the Methylomonas protein. Therefore, it would appear that only the residues involved in catalysis are conserved between BLF1 and CWO84_RS12955. Whilst such residues are often highly conserved across homologous enzymes, their conservation alone does not necessarily indicate preservation of substrate specificity or precise target recognition, as variations in the surrounding residues and active-site architecture can modulate enzyme specificity (Ribeiro et al., 2020 ▸).

4. Discussion

Despite low sequence identity, bacterial deamidases such as CNF1 and BLF1 have a similar fold and share a conserved cysteine–histidine dyad as part of their catalytic machinery, illustrating the preservation of core enzymatic mechanisms across divergent proteins (Buetow & Ghosh, 2003 ▸; Mobbs et al., 2022 ▸). However, sequence differences in their substrate-recognition regions allow these enzymes to target distinct host proteins, as observed across related toxin families (Barbieri et al., 2002 ▸). More broadly, this separation between catalytic conservation and substrate specificity is a common feature of enzyme evolution, as exemplified by cysteine-dependent enzyme families such as papain-like proteases, in which the catalytic arrangement is preserved despite variation in the substrate-binding regions (Rawlings & Barrett, 1994 ▸; Storer & Ménard, 1994 ▸). Equivalent findings can be seen between different members of the papain-like cysteine protease family (Petushkova et al., 2022 ▸). In contrast, comparing the serine proteases subtilisin and trypsin/chymotrypsin, a different pattern emerges, with convergent evolution giving rise to evolutionarily unrelated enzymes with distinct overall folds but retaining analogous catalytic mechanisms (Denesyuk et al., 2024 ▸; Wallace et al., 1996 ▸). These examples demonstrate that preservation of catalytic geometry and mechanistic strategy can be maintained even in the absence of shared structural scaffolds or evolutionary ancestry, highlighting the strong selective constraints acting on efficient catalytic solutions across diverse protein folds.

The broad phylogenetic distribution of BLF1-like deamidases suggests that their evolution may involve vertical inheritance or horizontal gene transfer (Gal-Mor & Finlay, 2006 ▸; Hacker & Kaper, 2000 ▸; Juhas et al., 2009 ▸). One possible explanation is vertical inheritance from an ancestral bacterial lineage followed by extensive divergence during speciation. Under this condition, an ancestral deamidase gene would have been retained in multiple descendant lineages, while sequence variation accumulated over evolutionary time. The preservation of catalytic residues and active-site architecture despite low overall sequence identity is consistent with this scenario and indicates strong selective pressure to maintain enzymatic function. The identification of BLF1-like proteins in phylogenetically diverse bacteria, including Burkholderia, Xanthomonas and Methylomonas, supports the possibility that the deamidase fold represents an ancient enzymatic scaffold that is retained across divergent bacterial taxa.

Horizontal gene transfer may also have contributed to the current distribution of BLF1-like deamidases. In bacteria, mobile genetic elements such as plasmids, transposons and genomic islands facilitate the transfer of adaptive and virulence-associated genes across species boundaries, thereby accelerating bacterial evolution and niche adaptation (Gal-Mor & Finlay, 2006 ▸; Hacker & Kaper, 2000 ▸; Juhas et al., 2009 ▸). The presence of BLF1-like proteins in distantly related bacterial species occupying overlapping ecological niches may therefore reflect lateral acquisition events. This possibility is particularly intriguing for Methylomonas sp. Kb3, a methanotroph that is not known to be associated with BLF1-like toxins, suggesting that the gene encoding this BLF1-like protein may have been acquired via environmental gene exchange. Comparable patterns have been observed for virulence factors such as CNF1, which is encoded within pathogenicity island II (PAI II) of uropathogenic E. coli, one of several pathogenic islands identified in this strain (Dobrindt et al., 2002 ▸), illustrating how horizontally transferred genomic regions can disseminate functionally important toxin genes across bacterial lineages.

Methylomonas and related aerobic methanotrophs are detected in plant-associated environments such as the phyllosphere and rhizosphere, particularly in methane-rich habitats, indicating their ability to inhabit plant surfaces. However, to date no evidence exists for intracellular replication of Methylomonas within plant cells (Iguchi et al., 2015 ▸; Knief, 2015 ▸). Consequently, despite the conservation of catalytic geometry in CWO84_RS12955 that parallels BLF1, current evidence does not support eIF4A as a physiologically relevant target in this bacterium. This highlights that although structural conservation is strong, functional context in the native organism can differ markedly from that in pathogenic relatives.

While crystallographic and structural analyses provide compelling evidence that CWO84_RS12955 from Methylomonas preserves key elements of the catalytic geometry required for deamidase activity, several fundamental questions remain unresolved. It is unknown whether CWO84_RS12955 is expressed under physiological or environmental conditions relevant to the lifestyle of Methylomonas, how its expression is regulated or which signals control its production. The genus Methylomonas consists of strictly aerobic, obligate methanotrophs that use methane and methanol as their sole energy sources in freshwater sediment and related ecosystems (Tikhonova et al., 2023 ▸). While some bacteria use specialized secretion systems, such as type III secretion systems, to deliver effectors into the host cytosol during pathogenic interactions (Coburn et al., 2007 ▸), no such systems have been described for Methylomonas. Therefore, the biological context and cellular role of CWO84_RS12955 remain unclear.

B. pseudomallei is an intracellular pathogen and BLF1 contributes directly to host infection and virulence, as shown by its inhibition of eIF4A and attenuation of virulence in the absence of the blf1 gene (Bzdyl et al., 2022 ▸; Cruz-Migoni et al., 2011 ▸). In contrast, no evidence exists that Methylomonas species adopt an intracellular lifestyle in plants. Consequently, the role of CWO84_RS12955 in the biology of the microbe remains unresolved. Addressing these questions will clarify whether CWO84_RS12955 has an intracellular role within Methylomonas or whether it functions as an effector protein to control the biology of some of the species which interact with Methylomonas. Such insights will provide a strong rationale for future studies focused on biochemical validation, regulatory analysis and functional characterization of this protein.

5. Conclusion

The crystal structure of CWO84_RS12955 from Methylomonas sp. Kb3 suggests that it acts as a BLF1-like deamidase with preservation of the catalytic cysteine–histidine architecture despite marked sequence divergence. Although the canonical LSGC motif of BLF1 is replaced by MDGC in CWO84_RS12955, the active-site geometry remains strongly conserved. Structural comparison of CWO84_RS12955 and the BLF1C94S–eIF4A complex indicates that residues implicated in substrate recognition by BLF1 are not conserved to provide a framework for future biochemical characterization of its enzymatic activity and substrate specificity.

Supplementary Material

PDB reference: putative BLF1-like deamidase from Methylomonas sp. Kb3, 9vfk

Supplementary Figures. DOI: 10.1107/S2053230X2600868X/rf5051sup1.pdf

f-82-00336-sup1.pdf (390.6KB, pdf)

Acknowledgments

The authors would like to acknowledge Universiti Kebangsaan Malaysia and the Malaysia Genome and Vaccine Institute for providing the facilities and technical support necessary for this research. We also thank the staff of SPring-8, Hyogo, Japan for providing synchrotron beam time and technical assistance during X-ray diffraction data collection. The authors also utilized ChatGPT (OpenAI) to assist with improving the language, grammar and readability of the manuscript. All scientific interpretations, analyses and conclusions presented in this work were reviewed and verified by the authors.

Funding Statement

This work was supported by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (FRGS), grant No. FRGS/1/2019/STG05/UKM/02/11.

Conflict of interest

The authors declare that there are no conflicts of interest.

Data availability

The atomic coordinates and structure factors for the crystal structure reported in this study have been deposited in the Protein Data Bank (PDB) under accession code 9vfk. All other data supporting the findings of this study are provided within the article and its supporting information or are available from the corresponding authors upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: putative BLF1-like deamidase from Methylomonas sp. Kb3, 9vfk

Supplementary Figures. DOI: 10.1107/S2053230X2600868X/rf5051sup1.pdf

f-82-00336-sup1.pdf (390.6KB, pdf)

Data Availability Statement

The atomic coordinates and structure factors for the crystal structure reported in this study have been deposited in the Protein Data Bank (PDB) under accession code 9vfk. All other data supporting the findings of this study are provided within the article and its supporting information or are available from the corresponding authors upon reasonable request.

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