Phosphopantetheine adenylyltransferase (PPAT), a key enzyme in the universal Coenzyme A biosynthetic pathway, is essential for cellular metabolism. However, the adaptive mechanisms of PPAT in psychrophilic (cold-adapted) organisms remain poorly understood. Here, we characterize PPAT from the psychrophilic methanotroph Methylocapsa palsarum (MpaPPAT). Sequence analysis identified a unique five-amino-acid insertion (SCRLS) within a surface-exposed loop, a feature conserved among psychrophilic homologues. To investigate its function, we determined the crystal structures of wild-type (WT) MpaPPAT and a loop-deletion mutant (MpaPPAT(Δ67-71)) and performed comparative biochemical analyses. Structurally, MpaPPAT forms a dimer-of-trimers hexamer. Biochemically, WT MpaPPAT maintains high catalytic activity at low temperatures (10-20 °C), whereas the MpaPPAT(Δ67-71) mutant exhibits impaired cold activity. The mutant structure reveals that the deletion of the distant surface loop induces a long-range allosteric change, resulting in a dual impairment: 1) a stabilization and rigidification ("clamping") of the central α-helix 4 (H4) at the hexameric core interface, and 2) a dramatic shift in the central pore's electrostatic potential from positive (WT) to negative (mutant). Our findings reveal that the SCRLS insertion is a critical allosteric modulator that provides a sophisticated dual mechanism for enzymatic cold adaptation. It maintains the conformational flexibility of the hexameric core, preventing the "clamping" effect, and simultaneously ensures a positively charged central channel to electrostatically steer negatively charged substrates (ATP and phosphopantetheine) into the active site, thereby overcoming the kinetic challenges of a low-temperature environment.

Orotidine-5'-phosphate decarboxylase (OMP decarboxylase; ODCase) catalyzes a key step in de novo pyrimidine biosynthesis by converting orotidine-5'-monophosphate to uridine-5'-monophosphate. Despite its essential metabolic role, the structural basis underlying substrate recognition and binding dynamics in ODCase from Fusobacterium nucleatum (FnODCase) remains poorly understood. In the present study, we determined the crystal structure of FnODCase at 1.76 Å resolution in the P 1 21 1 space group, with Rwork and Rfree values of 0.21 and 0.23, respectively. To investigate the ligand specificity of FnODCase, we performed isothermal titration calorimetry using three nucleotides that share a pyrimidine ring: cytidine 5'-monophosphate, thymidine 5'-monophosphate, and uridine 5'-monophosphate. The ITC data showed that FnODCase selectively recognized only UMP, confirming its high specificity for the uridine moiety. Notably, structural comparisons between the apo form and UMP-bound models revealed potential conformational changes in the loop regions surrounding the active site. These structural insights are expected to inform the future development of selective antimicrobial agents against F. nucleatum.

Antarctic microorganisms experience persistent subzero temperatures and repeated freeze-thaw cycles that require specialized mechanisms for survival. Although transcriptomic studies have identified numerous cold-responsive genes, many remain annotated as hypothetical proteins with unknown functions. In this study, we investigated PL002-1792, a strongly upregulated hypothetical protein from Antarctic Flavobacterium sp. PL002 identified under severe cold stress (-20 °C versus -6 °C; log2 fold change = 5.61, adjusted p = 1.89 × 10-138). Sequence analysis revealed a 402-amino-acid protein containing a predicted Sec/SPII lipoprotein signal peptide. AlphaFold3 prediction generated a high-confidence structural model (pTM = 0.96) with an elongated β-sheet-rich architecture resembling bacterial ice-binding proteins. Comparative analysis with the ice-binding protein from Flavobacterium frigoris (FfIBP) identified conserved glycine-rich and TXT-like motifs associated with putative ice-binding surfaces. Recombinant PL002-1792 was expressed in Escherichia coli, recovered from inclusion bodies, and successfully refolded into a predominantly β-sheet-rich conformation as confirmed by circular dichroism spectroscopy. Functional assays demonstrated moderate ice recrystallisation inhibition activity, reducing relative ice crystal mean grain size to approximately 90% of the control, and significantly enhanced freeze-thaw survival, with recombinant cells retaining 67% viability after three freeze-thaw cycles compared with 27% for the empty-vector control. These findings identify PL002-1792 as a novel antifreeze-like protein and highlight the utility of structure-guided approaches for uncovering previously uncharacterized cold-adaptation mechanisms in polar microorganisms.

Class III aminotransferases represent a structurally and functionally unique subgroup. However, the contribution of specific loop elements to their active-site architecture and cofactor-dependent structural transitions remain underexplored. We elucidated the structural framework underlying the catalytic function of class III fold Pyridoxal 5′-phosphate-dependent aminotransferase from the Antarctic bacterium Hymenobacter sp. PAMC 26554 (HyAT), and report its crystal structure at 2.31 Å resolution. The structure revealed a canonical class III fold organized as a functional homotetramer. Structural analysis identified a proline-containing motif (P-x-P) within the α10-α11 loop, which induces intrinsic disorder at the active-site entrance in the apo-form and revealed that a cooperative disorder-to-order transition is requisite for active-site assembly upon cofactor binding. We propose that this flexible loop region may be involved in modulating substrate access. Notably, this proline motif was conserved in homologs from Hyperthermophiles, despite the cold-adapted nature of HyAT. This convergence implies a common evolutionary strategy where the geometric constraints of proline are exploited to decouple local active-site dynamics from global scaffold stability, thereby addressing the stability–activity trade-off across diverse thermal environments. Our findings provide new molecular insights into the structural dynamics of class III aminotransferases and highlight evolutionary strategies for tuning enzyme flexibility in extreme environments.

Cold-active lipolytic enzymes enable low-temperature biocatalysis, but remain underexplored in Antarctic actinomycetes. Here, we report the discovery and first-step characterization of a CALB-like cold-active lipolytic enzyme (PanLip) from Pseudonocardia antarctica. Sequence and structure analyses revealed a canonical α/β-hydrolase fold with a conserved Ser–Asp–His triad and short helical elements around the pocket reminiscent of CALB’s α5/α10 lid. Mature PanLip was expressed primarily as inclusion bodies in E. coli; an N-terminally truncation (PanLipΔN) improved solubility and PanLipΔN was purified by Ni–NTA. Far-UV CD confirmed a folded α/β architecture. PanLipΔN favored short-chain substrates (p-NPA, kcat/KM = 2.4 × 105 M−1·s−1) but also showed measurable hydrolytic activity toward natural triglycerides, consistently with a lipase-family esterase. The enzyme showed an activity optimum near 25 °C and pH 8.0. The enzyme tolerated low salt (maximal at 0.1 M NaCl), mild glycerol, and selected organic solvents (notably n-hexane), but was inhibited by high salt, Triton X-100, and SDS. AlphaFold predicted high local confidence for the catalytic core; DALI placed PanLip closest to fungal lipases (AFLB/CALB). Temperature-series MD and CABS-flex indicated enhanced surface breathing and flexible segments adjacent to the active site—including a region topologically matching CALB α10—supporting a flexibility-assisted access mechanism at low temperature. Structure-based MSAs did not support a cold adaptation role for the reported VDLPGRS motif. Taken together, these findings position PanLip as a promising cold-active catalyst with CALB-like access control and potential for low-temperature biocatalysis.

Streptococcus salivarius K12 (SAL) is an oral probiotic used to treat or prevent oral infections caused by human pathogens. SAL produces at least three antimicrobials to exert its antimicrobial activity, namely, salivaricin A and salivaricin B, and the newly identified salivabactin. Salivabactin production is catalyzed by a polyketide/non-ribosomal peptide synthase hybrid biosynthetic gene cluster (BGC), termed as sar-BGC. The sar-BGC expression and salivabactin production are transient during SAL growth in vitro and in vivo, which may negatively impact SAL probiotic efficacy. To understand the molecular basis for transient sar-BGC expression, we assessed the impact of environmental pH on sar-BGC expression. We found that environmental acidification is a critical factor in promoting salivabactin antimicrobial activity and production by inducing sar-BGC expression. We further showed that acidic pH directly influences the quorum-sensing system that controls sar-BGC expression. During environmental acidification, SAL cytosol is acidified, which is sensed by a pH-sensitive histidine switch in the cytosolic transcription regulator, NrpR. The protonation of histidine during cytosolic acidification promotes high-affinity interactions between NrpR and its cognate intercellular signaling peptide, NIP, which leads to upregulation of sar-BGC expression. Collectively, our results indicate that SAL uses a sophisticated regulatory mechanism to orchestrate salivabactin production in an environment that is conducive to its antimicrobial activity. IMPORTANCE:Probiotic bacteria are important tools in combating bacterial infections. Probiotics exert their antimicrobial activity via several mechanisms, including antimicrobial production. However, discrepancies exist between the in vitro and in vivo efficacies of probiotics in inhibiting pathogen growth. Understanding the host and environmental factors that influence antimicrobial production and activity is critical for improving probiotic efficacy. In this study, we showed that the antimicrobial salivabactin produced by human oral probiotic Streptococcus salivarius K12 is active at acidic pH. We further elucidated the molecular mechanism by which S. salivarius coordinates salivabactin production in concert with environmental acidification, thereby maximizing salivabactin antimicrobial activity.

BACKGROUND:Adenine phosphoribosyltransferase (APRT) is an enzyme that facilitates adenosine monophosphate (AMP) biosynthesis by transferring a phosphoribosyl group to adenine using phosphoribosyl pyrophosphate as a donor. While the human enzyme is well characterized, structural insights into bacterial APRTs remain limited. Fusobacterium nucleatum is associated with periodontal disease, yet its APRT enzyme (FnAPRT) has not been structurally investigated. OBJECTIVE:This study aimed to examine the crystal structure of FnAPRT and ligand-induced conformational changes to understand its enzymatic and substrate recognition mechanisms. METHODS:The FnAPRT protein was heterologously expressed in Escherichia coli, followed by initial purification using nickel-charged affinity resin chromatography and further purification through size-exclusion chromatography. The FnAPRT structure was resolved using X-ray crystallography and compared with that of E. coli APRT (EcAPRT), exhibiting the highest amino acid sequence similarity among bacterial APRT structures. RESULTS:AMP and phosphate (PO4) were observed in the active site of FnAPRT. Significant differences in ligand positioning were observed between the AMP-PO4-bound structures of FnAPRT and EcAPRT. Structural shifts induced by AMP-PO4 binding were detected. The Arg78 and Lys82 residues from the alternate subunit occupied the PO4 site in the absence of ligands, but they interacted with PO4 upon AMP-PO4 binding. Structural comparison of the AMP-PO4-bound FnAPRT with that of the adenine-bound EcAPRT highlighted variations in the adenine-binding site and associated structural changes. DISCUSSION:Structural comparison of the AMP-PO4-bound FnAPRT with that of the adeninebound EcAPRT highlighted variations in the adenine-binding site and the associated structural changes. CONCLUSION:The AMP-PO4-bound FnAPRT exhibited distinct ligand-binding modes despite sharing a high sequence similarity with EcAPRT. The structures demonstrated ligand movement during bacterial APRT reactions.

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity on them remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and lake environments, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels (∼120 μS/cm), comparable to values commonly found in pristine terrestrial and aquatic environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha , significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by “ Ca. Nitrosotenuis” of the Nitrosopumilaceae family, was stimulated by high salinity (∼760 μS/cm). In ammonium-fed lake microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low (∼170 μS/cm) and high salinity (∼850 μS/cm) conditions. In the presence of allylthiourea, a bacterial nitrification inhibitor, AOA were sensitive to low salinity in both soil and lake microcosms. Consistently, pure culture studies revealed marked growth inhibition of AOA, especially members of Nitrosopumilaceae , under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Comparative genomic analyses with AOB and comammox, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of sophisticated osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study highlights hypoosmolarity as a key factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and aquatic environments that are increasingly affected by intensified water cycles due to climate change. ### Competing Interest Statement The authors have declared no competing interest. Ministry of Science and ICT, https://ror.org/01wpjm123, 2021R1A2C3004015, RS-2023-00213601 Ministry of Education, 2020R1A6A1A06046235 FWF Austrian Science Fund, 10.55776/COE7

Protein purification is essential for the isolation of specific proteins from mixtures. Conventional affinity tags have advanced recombinant protein purification. However, their reliance on costly resins and complex procedures often limits scalability and affordability. In this study, we identified three ice-binding domains (CoIBD1, CoIBD2, and CoIBD3) in Candidatus Cryosericum odellii SMC5 to evaluate their potential as protein purification tags. These domains exhibited hyperactive ice-binding properties, including high thermal hysteresis and ice recrystallization inhibition activities; additionally, they bound to multiple ice planes, enabling efficient attachment to ice surfaces. Through sequence and structural analyses, we engineered an enhanced variant that retained these ice-binding traits while achieving improved thermal and chemical stability: eCoIBD1. We then used eCoIBD1 as a fusion tag to develop the Ice Affinity Purification (IAP) system and evaluated its performance with GFP as a model protein. The IAP system achieved 87 % purity after two purification rounds, recovering 29 % of the initial protein from the crude extract. Consistent performance was observed in the presence of additives such as dithiothreitol and glycerol. The IAP system provides a cost-effective, environmentally friendly alternative to traditional methods by leveraging ice as a renewable binding medium, thereby eliminating the need for expensive resins or regeneration steps.

We report the complete genome sequence of Rahnella sp. PAMC25559, isolated from Zugspitz glacier, Austria. The complete genome assembly was generated, consisting of one chromosome and two putative plasmid sequences. The genome includes biosynthetic gene clusters potentially related to cold adaptation and plant growth-promoting activity.

Adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) is one of the key enzymes that participate in the biosynthesis of cobalamin, specifically lining the lower ligand 5,6-dimethylbenzimidazole in the α-position of cyclic tetrapyrrolidine. During this process, CobU exhibits two distinct activities: kinase and nucleotidyl transferase, using two nucleoside triphosphates. A structural study of CobU from Salmonella typhimurium showed that guanosine triphosphate binding induces a conformational rearrangement of helix 2. This rearrangement decreases the distance between the phosphate binding loop (P-loop) and helix 2, which is important for the subsequent guanylylation step of the reaction. However, these findings provide only partial insights into the mechanism of CobU at the structural level, and the precise molecular details of this mechanism have not yet been studied. As a first step towards elucidating the molecular mechanisms and sequence of events involved in the phosphorylation and guanylylation steps, we report the high-resolution crystal structures of phosphorylated -MpaCobU (1.8 Å), the C91S mutant (1.5 Å), the guanosine diphosphate complex (1.9 Å), and the adenosylcobinamide-phosphate complex (2.6 Å) from Methylocapsa palsarum for the first time. High-resolution structures revealed the crucial elements governing the catalytic steps of MpaCobU, thereby contributing to understanding the catalytic mechanism of CobU at the molecular level.

Epoxide hydrolases (EHs), which catalyze the transformation of epoxides to diols, are present in many eukaryotic and prokaryotic organisms. They have recently drawn considerable attention from organic chemists owing to their application in the semisynthesis of enantiospecific diol compounds. Here, we report the crystal structures of BoEH from Bosea sp. PAMC 26642 and CaEH from Caballeronia sordidicola PAMC 26510 at 1.95 and 2.43 Å resolution, respectively. Structural analysis showed that the overall structures of BoEH and CaEH commonly possess typical α/β hydrolase fold with the same ring-opening residues (Tyr-Tyr) and conserved catalytic triad residues (Asp-Asp-His). However, the two enzymes were found to have significantly different sequence compositions in the cap domain region, which is involved in the formation of the substrate-binding site in both enzymes. Enzyme activity assay results showed that BoEH had the strongest activity toward the linear aliphatic substrates, whereas CaEH had a higher preference for aromatic- and cycloaliphatic substrates. Computational docking simulations and tunnel identification revealed important residues with different substrate-binding preferences. Collectively, structure comparison studies, together with ligand docking simulation results, suggested that the differences in substrate-binding site residues were highly correlated with substrate specificity.

Salinity poses a significant challenge to plant growth and crop productivity by adversely affecting crucial processes, including photosynthesis. Efforts to enhance abiotic stress tolerance in crops have been hindered by the trade-off effect, where increased stress resistance is accompanied by growth reduction. In this study, we identified and characterized a plastocyanin gene (PaPC) from the Antarctic moss Polytrichastrum alpinum, which enhanced photosynthesis and salt stress tolerance in Arabidopsis thaliana without compromising growth. While there were no differences in growth and salt tolerance between the wild type and Arabidopsis plastocyanin genes (AtPC1 and AtPC2)-overexpressing plants, PaPC-overexpressing plants demonstrated superior photosynthetic efficiency, increased biomass, and enhanced salt tolerance. Similarly, PaPC-overexpressing rice plants exhibited improved yield potential and photosynthetic efficiency under both normal and salt stress conditions. Key amino acid residues in PaPC responsible for this enhanced functionality were identified, and their substitution into AtPC2 conferred improved photosynthetic performance and stress tolerance in Arabidopsis, tobacco, and tomato. These findings not only highlight the potential of extremophiles as valuable genetic resources but also suggest a photosynthesis-based strategy for developing stress-resilient crops without a growth penalty.

Ice accumulation and proliferation adversely affect the activities of various residential, commercial, and polar research stations. Although significant efforts are devoted to preventing ice adhesion to various surfaces by developing various anti-icing coatings, it is still necessary to enhance overall performance and durability. Herein, a facile approach is proposed for fabricating an icephobic coating on an aluminum 6061 (Al) substrate, by coating a poly(dimethylsiloxane) (PDMS)/ poly(tetrafluoroethylene) (PTFE) composite through a spin-coating method, followed by sprinkling of SiO2 nanoparticles (NPs). Crosslinker/binder-free adhesion between PDMS and PTFE is achieved by utilizing secondary-induced electrostatic dipole-dipole interactions, these interactions are supported by density functional theory (DFT) calculations as well as structural studies. Moreover, the controlled addition of PTFE powder to PDMS improves the water-repellency, mechanical strength, and surface roughness of the coating. The self-formation of the superhydrophobic state of the PDMS/PTFE composite is achieved by sprinkling SiO2 NPs. The sprinkled SiO2 NPs are protected by the PDMS/PTFE composite, which serves as a stress concentrator to achieve low ice adhesion. Furthermore, freezing at low temperatures can be delayed by controlling the heat flow rate, interfacial contact area, and surface texture. This indicates the feasibility of the proposed method for various promising anti-icing applications.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing–thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.

Sulfurtransferases transfer of sulfur atoms from thiols to acceptors like cyanide. They are categorized as thiosulfate sulfurtransferases (TSTs) and 3-mercaptopyruvate sulfurtransferases (MSTs). TSTs transfer sulfur from thiosulfate to cyanide, producing thiocyanate. MSTs transfer sulfur from 3-mercaptopyruvate to cyanide, yielding pyruvate and thiocyanate. The present study aimed to isolate and characterize the sulfurtransferase FrST from Frondihabitans sp. PAMC28461 using biochemical and structural analyses. FrST exists as a dimer and can be classified as a TST rather than an MST according to sequence-based clustering and enzyme activity. Furthermore, the discovery of activity over a wide temperature range and the broad substrate specificity exhibited by FrST suggest promising prospects for its utilization in industrial applications, such as the detoxification of cyanide.

Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. Here, CESS-1 was revealed to display hydrolytic activities toward penicillins (penicillin G and ampicillin) and cephalosporins (cephalexin, cefaclor, and cefotaxime), while its activity toward carbapenems (imipenem and meropenem) was negligible. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward the three β-lactam antibiotics were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were calculated to be 1249.6 s−1, 204.3 s−1, and 69.8 s−1, respectively, with the accompanying KM values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively. Remarkably, CESS-1 discriminates cefaclor and cephalexin with only one structural difference: –Cl (cefaclor) and –CH3 (cephalexin) at C3. According to structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, and ampicillin, the cooperative positional changes of the R1 side chain of substrates and its contacting β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics.

Probiotic supplements are suggested to promote human health by preventing pathogen colonization. However, the mechanistic bases for their efficacy in vivo are largely uncharacterized. Here using metabolomics and bacterial genetics, we show that the human oral probiotic Streptococcus salivarius K12 (SAL) produces salivabactin, an antibiotic that effectively inhibits pathogenic Streptococcus pyogenes (GAS) in vitro and in mice. However, prophylactic dosing with SAL enhanced GAS colonization in mice and ex vivo in human saliva. We showed that, on co-colonization, GAS responds to a SAL intercellular peptide signal that controls SAL salivabactin production. GAS produces a secreted protease, SpeB, that targets SAL-derived salivaricins and enhances GAS survival. Using this knowledge, we re-engineered probiotic SAL to prevent signal eavesdropping by GAS and potentiate SAL antimicrobials. This engineered probiotic demonstrated superior efficacy in preventing GAS colonization in vivo. Our findings show that knowledge of interspecies interactions can identify antibiotic- and probiotic-based strategies to combat infection.

Porphyromonas gingivalis is a major pathogenic oral bacterium that is responsible for periodontal disease. It is linked to chronic periodontitis, gingivitis and aggressive periodontitis. P. gingivalis exerts its pathogenic effects through mechanisms such as immune evasion and tissue destruction, primarily by secreting various factors, including cysteine proteases such as gingipain K (Kgp), gingipain R (RgpA and RgpB) and PrtH (UniProtKB ID P46071). Virulence proteins comprise multiple domains, including the pro-peptide region, catalytic domain, K domain, R domain and DUF2436 domain. While there is a growing database of knowledge on virulence proteins and domains, there was no prior evidence or information regarding the structure and biological function of the well conserved DUF2436 domain. In this study, the DUF2436 domain of PrtH from P. gingivalis (PgDUF2436) was determined at 2.21 Å resolution, revealing a noncanonical β-jelly-roll sandwich topology with two antiparallel β-sheets and one short α-helix. Although the structure of PgDUF2436 was determined by the molecular-replacement method using an AlphaFold model structure as a template, there were significant differences in the positions of β1 between the AlphaFold model and the experimentally determined PgDUF2436 structure. The Basic Local Alignment Search Tool sequence-similarity search program showed no sequentially similar proteins in the Protein Data Bank. However, DaliLite search results using structure-based alignment revealed that the PgDUF2436 structure has structural similarity Z-scores of 5.9-5.4 with the C-terminal domain of AlgF, the D4 domain of cytolysin, IglE and the extracellular domain structure of PepT2. This study has elucidated the structure of the DUF2436 domain for the first time and a comparative analysis with similar structures has been performed.

Heavy metals, including mercury, are non-biodegradable and highly toxic to microorganisms even at low concentrations. Understanding the mechanisms underlying the environmental adaptability of microorganisms with Hg resistance holds promise for their use in Hg bioremediation. We characterized Gbs MerA, a mercury reductase belonging to the mercury-resistant operon of Gelidibacter salicanalis PAMC21136, and found its maximum activity of 474.7 µmol/min/mg in reducing Hg +2 . In the presence of Ag and Mn, the enzyme exhibited moderate activity as 236.5 µmol/min/mg and 69 µmol/min/mg, respectively. Gbs MerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of Gbs MerA and structural comparison with homologues indicated that Gbs MerA contains residues, Tyr437´ and Asp47, which may be responsible for metal transfer at the si -face by providing a hydroxyl group (−OH) to abstract a proton from the thiol group of cysteine. The complex structure with NADPH indicated that Y174 in the re -face can change its side chain direction upon NADPH binding, indicating that Y174 may have a role as a gate for NADPH binding. Moreover, the heterologous host expressing Gbs MerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking Gbs MerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by Gbs MerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.