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MltG, a membrane-bound lytic transglycosylase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosylation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus.

Background: Recent studies have elucidated the quorum-sensing mechanisms, biofilm formation, inter-pathogen interactions, and genes related to oral pathogens. This review aims to explore the recent expansion of drug targets against oral pathogens and summarize the current research on novel antibiotic substances derived from marine organisms that target oral pathogens. Methods: A comprehensive literature review summarized the novel mechanisms pertaining to quorum-sensing signal transmission systems, biofilm formation, and metabolite exchange in oral pathogens. The amino acid sequences of the 16 proteins identified as potential drug targets were systematically classified and compared across various oral microorganisms. Results: Through a literature review, we identified nine studies researching quorum sensing signaling inhibitors targeting oral pathogens. A comparison of the amino acid sequences of 16 potential drug targets in oral microorganisms revealed significant differences between oral pathogens and beneficial oral symbiotic microorganisms. These findings imply that it is possible to design drugs that can bind more selectively to oral pathogens. Conclusion: By summarizing the results of recent research on the signaling mechanisms that cause pathogenicity, new drug targets against oral pathogens were proposed. Additionally, the current status of developing new antibiotics for oral pathogens using recently developed quorum sensing inhibitors and natural products derived from marine organisms was introduced. Consequently, marine natural products can be used to develop drugs targeting new proteins in oral pathogens.

This study characterized cytochrome P450 enzyme CYP154C7 from Streptomyces sp. PAMC26508, emphasizing its capability to hydroxylate steroids, especially at the 16α-position. The enzymatic assay of CYP154C7 demonstrated effective conversion across a pH range of 7.2–7.6, with optimal activity at 30 °C in the Pdx/PdR plus NADH system. Kinetic analysis on most converted steroids (androstenedione and adrenosterone) was performed which shows a greater affinity for androstenedione (Km, 11.06 ± 1.903 μM; Vmax, 0.0062 ± 0.0002 sec−1) compared to adrenosterone (Km, 34.50 ± 6.2 μM; Vmax, 0.0119 ± 0.0007 sec−1). A whole-cell system in Escherichia coli, overexpressing recombinant CYP154C7, achieved substantial conversion for steroids, indicating that CYP154C7 can also be used as a potential whole-cell biocatalyst. To gain structural insights, homology models of CYP154C7 and its homologs were constructed using CYP154C5 (PDB ID: 6TO2), refined, validated, and used for docking studies. Comparative docking analysis suggests that lysine (K236) in the active site and tyrosine (Y197) in the substrate access channel of CYP154C7 are crucial for substrate selectivity and catalytic efficiency. This study suggests that CYP154C7 could be a promising candidate for developing modified steroids, providing valuable insights for protein engineering to design commercially useful CYP steroid hydroxylases with diverse substrate specificities.

Artemia is a crustacean genus belonging to the order Anostraca in the class Branchiopoda and lives in inland hypersaline lakes. Among the genus, A. franciscana is a valuable species as a fish food in the aquaculture industry or as an aquatic model organism for toxicity tests. However, genomic data for A. franciscana remains incomplete. In this study, high-quality genome assembly at the chromosome level of female A. franciscana was conducted by combining various sequencing and assembly technologies. The final A. franciscana assembled genome was 1.27 Gb in length, containing 21 chromosomal scaffolds (>10 Mb). The scaffold N50 was 45.3 Mb, with a complete BUSCO value of 91.0 %, thereby confirming that a high-quality genome was assembled. Gene annotation shows that the A. franciscana genome contained 67.26 % of repetitive sequences, and a total of 26,923 protein-coding genes were predicted. Among the 21 chromosome-scale scaffolds, chromosome 1 was identified as a sex chromosome Z. Additionally, five contigs of putative W chromosome fragments and the candidate sex-determining genes were suggested. Ten homeobox (Hox) genes were identified in A. franciscana on the chromosome 14, which were in two subclusters with a large gap. Hox gene organizations within 13 arthropods showed that four anostracans had conserved synteny. This study provides a new female Artemia genome with sex chromosome and the first complete genomic arrangement of the Hox cluster in Anostraca. This study will be a useful genomic and genetic reference for understanding the evolution and development of A. franciscana.

Abstract 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.

Abstract Inflammation is an essential defense mechanism in health; however, excessive inflammation contributes to the pathophysiology of several chronic diseases. Although anti-inflammatory drugs are essential for controlling inflammation, they have several side effects. Recent findings suggest that naturally derived compounds possess physiological activities, including anti-inflammatory, antifungal, antiviral, anticancer, and immunomodulatory activities. Therefore, this study aimed to investigate the anti-inflammatory effects and molecular mechanisms of 2,5,6-trimethoxy-p-terphenyl (TP1), extracted from the Antarctic lichen Stereocaulon alpinum, using in vitro models. TP1 treatment decreased the production of nitric oxide (NO) and reactive oxygen species (ROS) in LPS-stimulated Raw264.7 macrophages. Additionally, TP1 treatment significantly decreased the mRNA levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and the mRNA and protein levels of the pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Moreover, TP1 suppressed lipopolysaccharide-induced phosphorylation of the NF-κB and MAPK signaling pathways in Raw264.7 macrophages. Conclusively, these results suggest that TP1 ameliorates inflammation by suppressing the expression of pro-inflammatory cytokines, making it a potential anti-inflammatory drug for the treatment of severe inflammatory diseases.

Abstract Cladonia borealis is a lichen that inhabits Antarctica’s harsh environment. We sequenced the whole genome of a C. borealis culture isolated from a specimen collected in Antarctica using long-read sequencing technology to identify specific genetic elements related to its potential environmental adaptation. The final genome assembly produced 48 scaffolds, the longest being 2.2 Mbp, a 1.6 Mbp N50 contig length, and a 36 Mbp total length. A total of 10,749 protein-coding genes were annotated, containing 33 biosynthetic gene clusters and 102 carbohydrate-active enzymes. A comparative genomics analysis was conducted on six Cladonia species, and the genome of C. borealis exhibited 45 expanded and 50 contracted gene families. We identified that C. borealis has more Copia transposable elements and expanded transporters (ABC transporters and magnesium transporters) compared to other Cladonia species. Our results suggest that these differences contribute to C. borealis’ remarkable adaptability in the Antarctic environment. This study also provides a useful resource for the genomic analysis of lichens and genetic insights into the survival of species isolated from Antarctica.

Abstract Malignant melanoma is a fatal disease with an increasing global incidence. Despite numerous studies focused on anti-cancer drugs, a variety of side effects of cancer treatment remain challenging. Thus, there is a pressing need to identify novel anti-cancer agents with minimal cytotoxicity and side effects. DB3 (1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di[ethanoyl]dibenzofuran) is a member of the dibenzofuran family and is extracted from Ramalina terebrata (Antarctic lichen). We investigated if DB3 exerted an antitumor effect on B16F10 melanoma cells. The results revealed that DB3 exerted time- and dose-dependent reduction of cell viability by inducing apoptosis and significantly suppressing cell proliferation through cell cycle arrest in the G0/G1 phase in B16F10 melanoma cells. Additionally, DB3 impeded the migration and invasiveness of B16F10 cells. Subsequently, we observed that DB3 decreased the expression levels of Cdk4/Cyclin D1 and the phosphorylation of p38, JNK, ERK, and AKT. Furthermore, DB3 decreased melanoma tumor growth in a mouse tumor syngraft model. Based on these findings, we propose that DB3 possesses potential for use as an anti-cancer agent for melanoma treatment.

AbstractThe complete mitochondrial genome of Trematomus newnesi was sequenced using an Illumina platform. The 18,602 bp mitogenome contains 13 protein-coding genes, two rRNAs, and 23 tRNAs (tRNAMet is duplicated). The eight stop codons are TAA, TAG, CTT, GTA, AAT, ACT, AGG, and TTA. Two start codons ATG and GTG are present. The GC content is 44.4% and AT content is 55.6%. A phylogenetic tree was generated using 13 species from three families. The results showed that T. newnesi is closely related to Pagothenia borchgrevinki in Nototheniidae. This study provides fundamental data for further genetic evolutionary studies on T. newnesi.

Abstract 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 GbsMerA, 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. GbsMerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of GbsMerA and structural comparison with homologues indicated that GbsMerA 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 GbsMerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking GbsMerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by GbsMerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.

Abstract A great diversity of crustacean zooplankton found in inland and coastal waters produce embryos that settle into bottom sediments to form an egg bank. Embryos from these banks can remain dormant for centuries, creating a reservoir of genetic diversity. A large body of literature describes the ecological and evolutionary importance of zooplankton egg banks. However, literature on the physiological traits behind dormancy in crustacean zooplankton are limited. Most data on the physiology of dormancy comes from research on one species of anostracan, the brine shrimp, Artemia franciscana. Anoxia-induced dormancy in this species is facilitated by a profound and reversible acidification of the intracellular space. This acidification is accompanied by a reversible depletion of adenosine triphosphate (ATP). The present study demonstrates that acidification of the intracellular space also occurs in concert with a depletion of nucleoside triphosphates (NTPs) in the Antarctic copepod, Boeckella poppei. Like A. franciscana, the depletion of NTPs and acidification are rapidly reversed during aerobic recovery in B. poppei. These data provide the first comparative evidence that extreme dormancy under anoxia in crustacean zooplankton is associated with intracellular acidification and an ability to recover from the depletion of ATP.

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.

Thioredoxin (Trx) is essential in a redox-control system, with many bacteria containing two Trxs: Trx1 and Trx2. Due to a Trx system's critical function, Trxs are targets for novel antibiotics. Here, a 1.20 Å high-resolution structure of Trx2 from Acinetobacter baumannii (abTrx2), an antibiotic resistant pathogenic superbug, is elucidated. By comparing Trx1 and Trx2, it is revealed that the two Trxs possess similar activity, although Trx2 contains an additional N-terminal zinc-finger domain and exhibits more flexible properties in solution. Finally, it is shown that the Trx2 zinc-finger domain might be rotatable and that proper zinc coordination at the zinc-finger domain is critical to abTrx2 activity. This study enhances understanding of the Trx system and will facilitate the design of novel antibiotics.

PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure–function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix–turn–helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases.

Glaciimonas sp. PAMC28666, an extremophilic bacterium thriving in Antarctic soil and belonging to the Oxalobacteraceae family, represents the only complete genome of its genus available in the NCBI database. Its genome measures 5.2 Mb and comprises 4,476 genes (4,350 protein-coding and 72 non-coding). Phylogenetic analysis shows the strain PAMC28666 in a unique branch within the genus Glaciimonas, closely related to Glaciimonas alpine Cr9-12, supported by robust bootstrap values. In addition, strain PAMC28666 showed 77.08 and 23.3% ANI and DDH, respectively, with Glaciimonas sp. PCH181.This study focuses on how polar strain PAMC28666 responds to freeze–thaw conditions, Experimental results revealed a notable survival rate of 47.28% when subjected to a temperature of 15°C for a period of 10 days. Notably, two genes known to be responsive to cold stress, Trehalose 6-phosphate synthase (otsA) and Trehalose 6-phosphate phosphatase (otsB), exhibited increased expression levels as the temperature shifted from 25°C to 15°C. The upregulation of otsAB and the consequent synthesis of trehalose play pivotal roles in enhancing the cold resistance of strain PAMC28666, offering valuable insights into the correlation between trehalose production and adaptation to cold stress. Furthermore, research into this neglected cold-adapted variation, like Glaciimonas sp. PAMC28666, has the potential to shed light on how trehalose is produced in cold-adapted environments Additionally, there is potential to extract trehalose compounds from this strain for diverse biotechnological applications, including food and cosmetics, with ongoing research exploring its unique properties.

Abstract Parochlus steinenii is the only flying insect native to Antarctica. To elucidate the molecular mechanisms underlying its adaptation to cold environments, we conducted comparative genomic analyses of P. steinenii and closely related lineages. In an analysis of gene family evolution, 68 rapidly evolving gene families, involved in the innate immune system, unfolded protein response, DNA packaging, protein folding, and unsaturated fatty acid biosynthesis were detected. Some gene families were P. steinenii-specific and showed phylogenetic instability. Acyl-CoA delta desaturase and heat shock cognate protein 70 (Hsc70) were representative gene families, showing signatures of positive selection with multiple gene duplication events. Acyl-CoA delta desaturases may play pivotal roles in membrane fluidity, and expanded Hsc70 genes may function as chaperones or thermal sensors in cold environments. These findings suggest that multiple gene family expansions contributed to the adaptation of P. steinenii to cold environments.

In this study, Mesorhizobium sp. PAMC28654 was isolated from a soil sample collected from the polar region of Uganda. Whole-genome sequencing and comparative genomics were performed to better understand the genomic features necessary for Mesorhizobium sp. PAMC28654 to survive and thrive in extreme conditions and stresses. Additionally, diverse sequence analysis tools were employed for genomic investigation. The results of the analysis were then validated using wet-lab experiments. Genome analysis showed trace elements’ resistant proteins (CopC, CopD, CzcD, and Acr3), exopolysaccharide (EPS)-producing proteins (ExoF and ExoQ), and nitrogen metabolic proteins (NarG, NarH, and NarI). The strain was positive for nitrate reduction. It was tolerant to 100 mM NaCl at 15 °C and 25 °C temperatures and resistant to multiple trace elements (up to 1 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 1 mM ZnSO4·7H2O, 0.05 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 15 °C and 0.25 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 0.5 mM ZnSO4·7H2O, 0.01 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 25 °C). This research contributes to our understanding of bacteria’s ability to survive abiotic stresses. The isolated strain can be a potential candidate for implementation for environmental and agricultural purposes.

The methylenetetrahydrofolate dehydrogenase–cyclohydrolase (FolD) enzyme has a dual activity of N5,N10-methylenetetrahydrofolate dehydrogenase and cyclohydrolase. This enzyme plays a critical role in the chemical modification of tetrahydrofolate, which is an important coenzyme involved in the synthesis of DNA, RNA, and amino acids. Therefore, bacterial FolD has been studied as a potential drug target for the development of antibiotics. Here, we determined the crystal structure of FolD (PgFolD) from the oral pathogen Porphyromonas gingivalis at 2.05 Å resolution using the molecular replacement method. The crystal structure of PgFolD was successfully refined to a crystallographic R-factor of 21.4% (Rfree = 23.8%). The crystals belong to the space group of P4322 with the unit cell parameters of a = 110.7 Å, b = 110.7 Å, and c = 69.8 Å, containing one subunit in the asymmetric unit. Our analytical size-exclusion chromatography results indicated that PgFolD forms a stable dimer in solution. Additionally, structural and sequence comparison studies with previously known FolDs revealed that PgFolD has a different substrate-binding site residue composition. These findings provide valuable insights for the structure-based development of specific inhibitors against the Porphyromonas gingivalis pathogen.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Peptidoglycan digestion by murein-degrading enzymes is a critical process in bacterial cell growth and/or cell division. The membrane-bound lytic murein transglycosylase A (MltA) is a murein-degrading enzyme; it catalyzes the cleavage of the β-1,4-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans. Although substrate recognition and cleavage by MltA have been examined by previous structural and mutagenesis studies, the overall mechanism of MltA in conjunction with other functionally related molecules on the outer membrane of bacterial cells for peptidoglycan degradation has remained elusive. In this study, the crystal structure of MltA from the virulent human pathogen Acinetobacter baumannii is characterized and presented. The study indicated that MltA from A. baumannii forms homodimers via an extra domain which is specific to this species. Furthermore, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled based on the structural and biochemical analysis.

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Å resolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the β1 and β2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

Purpose: To quantitatively assess the angiographic features of uveitis associated with sarcoidosis on ultra-widefield fluorescein angiography (UWFA) and determine their correlations with clinical features. Design: A retrospective cohort study. Methods: Sixty-four eyes (64 patients) with sarcoidosis uveitis were included. On UWFA, presence of vasculitis, macular leakage, and optic disc leakage were assessed and features including peripheral ischemic area, vascular leakage area, and punched out lesions were quantitatively analyzed using FIJI (ImageJ2) and correlated with clinical features. Results: The mean peripheral ischemic area and leakage area were 0.0419 ± 0.113% and 0.0333 ± 0.0287% of the total retinal area, respectively. Macular and optic disc leakage were present in 18.8% and 59.4% of eyes, respectively. The average number of punched out lesions was 10.02 ± 21.95. Those changes were most abundant in the inferotemporal area. The presence of disc leakage correlated with all the other UWFA parameters (all r ≥ 0.260; all P ≤ 0.038). The leakage area correlated with vitreous cells, baseline and 6-month logMAR visual acuity, steroid dose and duration, erythrocyte sedimentation rate, and C-reactive protein (r = 0.472, 0.288, 0.321, 0.374, 0.250, 0.251, and 0.277; all P ≤ 0.46). Conclusions: This study quantitatively analyzed UWFA data in sarcoidosis uveitis. Angiographic changes were most frequent in the inferotemporal area. UWFA parameters correlated with one another and clinical variables. These quantitative imaging results warrant a subjective analysis of sarcoidosis uveitis.

Aspartate semialdehyde dehydrogenase (ASADH) catalyzes the biosynthesis of several essential amino acids, including lysine, methionine, and threonine, and bacterial cell components. Thus, ASADH is a crucial target for developing new antimicrobial agents that can potentially disrupt the biosynthesis of essential amino acids, thereby inhibiting the growth of pathogens. Herein, the crystal structures of ASADH obtained from Porphyromonas gingivalis (PgASADH, UniProtKB code A0A1R4DY25) were determined in apo- and adenosine-2′-5′-diphosphate (2′,5′-ADP)-bound complex forms at a resolution of 1.73 Å. The apo- and 2′,5′-ADP-complexed crystals of PgASADH belonged to the space groups of I212121 and C2221, respectively. Analytical size-exclusion chromatography showed a stable PgASADH dimer in a solution. Clustering analysis and structural comparison studies performed on PgASADH and previously known ASADHs revealed that ASADHs, including PgASADH, can be classified into three types depending on sequential and structural differences at the α-helical subdomain region. These findings provide valuable insights into developing structure-based species-specific new antibacterial drugs against the oral pathogen P. gingivalis.

Lipid II, the main component of the bacterial cell wall, is synthesized by the addition of UDP-N-acetylglucosamine to the UDP-N-acetylmuramic acid pentapeptide catalyzed by the glycosyltransferase MurG. Owing to its critical role in cell-wall biosynthesis, MurG is considered to be an attractive target for antibacterial agents. Although the Mur family ligases have been extensively studied, the molecular mechanism of the oligomeric scaffolding assembly of MurG remains unclear. In this study, MurG from Acinetobacter baumannii (abMurG), a human pathogen, was characterized and its hexameric crystal structure was unveiled; this is the first homo-oligomeric structure to be described in the MurG family and the Mur family. Homogeneous protein samples were produced for structural studies using size-exclusion chromatography, the absolute molecular mass was calculated via multi-angle light scattering, and protein–protein interactions were analyzed using the PDBePISA server. abMurG was found to form homo-oligomeric complexes in solution, which might serve as functional units for the scaffolding activity of MurG. Furthermore, analysis of this structure revealed the molecular assembly mechanism of MurG. This structural and biochemical study elucidated the homo-oligomerization mechanism of MurG and suggests a new potential antibiotic target on MurG.

The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM−1. The heme oxidation rate (k) was low (

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Abstract The expression of aminoglycoside-modifying enzymes represents a survival strategy of antibiotic-resistant bacteria. Aminoglycoside 2′-N-acetyltransferase [AAC(2′)] neutralizes aminoglycoside drugs by acetylation of their 2′ amino groups in an acetyl coenzyme A (CoA)-dependent manner. To understand the structural features and molecular mechanism underlying AAC(2′) activity, we overexpressed, purified, and crystallized AAC(2′) from Mycolicibacterium smegmatis [AAC(2′)-Id] and determined the crystal structures of its apo-form and ternary complexes with CoA and four different aminoglycosides (gentamicin, sisomicin, neomycin, and paromomycin). These AAC(2′)-Id structures unraveled the binding modes of different aminoglycosides, explaining the broad substrate specificity of the enzyme. Comparative structural analysis showed that the α4-helix and β8–β9 loop region undergo major conformational changes upon CoA and substrate binding. Additionally, structural comparison between the present paromomycin-bound AAC(2′)-Id structure and the previously reported paromomycin-bound AAC(6′)-Ib and 30S ribosome structures revealed the structural features of paromomycin that are responsible for its antibiotic activity and AAC binding. Taken together, these results provide useful information for designing AAC(2′) inhibitors and for the chemical modification of aminoglycosides.

Abstract Background Biodiesel and flavor compound production using enzymatic transesterification by microbial lipases provides mild reaction conditions and low energy cost compared to the chemical process. SGNH-type lipases are very effective catalysts for enzymatic transesterification due to their high reaction rate, great stability, relatively small size for convenient genetic manipulations, and ease of immobilization. Hence, it is highly important to identify novel SGNH-type lipases with high catalytic efficiencies and good stabilities. Results A promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from Halocynthiibacter arcticus was catalytically characterized and functionally explored. HaSGNH1 displayed broad substrate specificity that included tert-butyl acetate, glucose pentaacetate, and p-nitrophenyl esters with excellent stability and high efficiency. Important amino acids (N83, M86, R87, F131, and I173F) around the substrate-binding pocket were shown to be responsible for catalytic activity, substrate specificity, and reaction kinetics. Moreover, immobilized HaSGNH1 was used to produce high yields of butyl and oleic esters. Conclusions This work provides a molecular understanding of substrate specificities, catalytic regulation, immobilization, and industrial applications of a promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from H. arcticus. This is the first analysis on biodiesel and flavor synthesis using a cold-adapted halophilic SGNH-type lipase from a Halocynthiibacter species.

The complete genomes of Variovorax strains were analyzed and compared along with the genomes of Variovorax strains PAMC28711, PAMC28562, and PAMC26660, Antarctic isolates. The genomic information was collected from the NCBI database and the CAZyme database, and Prokka annotation was used to find the genes that encode for the trehalose metabolic pathway. Likewise, CAZyme annotation (dbCAN2 Meta server) was performed to predict the CAZyme family responsible for trehalose biosynthesis and degradation enzymes. Trehalose has been found to respond to osmotic stress and extreme temperatures. As a result, the study of the trehalose metabolic pathway was carried out in harsh environments such as the Antarctic, where bacteria Variovorax sp. strains PAMC28711, PAMC28562, and PAMC26660 can survive in extreme environments, such as cold temperatures. The trehalose metabolic pathway was analyzed via bioinformatics tools, such as the dbCAN2 Meta server, Prokka annotation, Multiple Sequence Alignment, ANI calculator, and PATRIC database, which helped to predict trehalose biosynthesis and degradation genes’ involvement in the complete genome of Variovorax strains. Likewise, MEGA X was used for evolutionary and conserved genes. The complete genomes of Variovorax strains PAMC28711, PAMC26660, and PAMC28562 are circular chromosomes of length (4,320,000, 7,390,000, and 4,690,000) bp, respectively, with GC content of (66.00, 66.00, and 63.70)%, respectively. The GC content of these three Variovorax strains is lower than that of the other Variovorax strains with complete genomes. Strains PAMC28711 and PAMC28562 exhibit three complete trehalose biosynthetic pathways (OtsA/OtsB, TS, and TreY/TreZ), but strain PAMC26660 only possesses one (OtsA/OtsB). Despite the fact that all three strains contain trehalose, only strain PAMC28711 has two trehalases according to CAZyme families (GH37 and GH15). Moreover, among the three Antarctica isolates, only strain PAMC28711 exhibits auxiliary activities (AAs), a CAZyme family. To date, although the Variovorax strains are studied for different purposes, the trehalose metabolic pathways in Variovorax strains have not been reported. Further, this study provides additional information regarding trehalose biosynthesis genes and degradation genes (trehalases) as one of the factors facilitating bacterial survival under extreme environments, and this enzyme has shown potential application in biotechnology fields.

Ornithine carbamoyltransferases (OTCs) are involved in the arginine deiminase (ADI) pathway and in arginine biosynthesis. Two OTCs in a pair are named catalytic OTC (cOTC) and anabolic OTC (aOTC). The cOTC is responsible for catalyzing the third step of the ADI pathway to catabolize citrulline into carbamoyl phosphate (CP), as well as ornithine, and displays CP cooperativity. In contrast, aOTC catalyzes the biosynthesis of citrulline from CP and ornithine in vivo and is thus involved in arginine biosynthesis. Structural and biochemical analyses were employed to investigate the CP cooperativity and unidirectional function of two sequentially similar OTCs (32.4% identity) named Ps_cOTC and Ps_aOTC from Psychrobacter sp. PAMC 21119. Comparison of the trimeric structure of these two OTCs indicated that the 80s loop of Ps_cOTC has a unique conformation that may influence cooperativity by connecting the CP binding site and the center of the trimer. The corresponding 80s loop region of in Ps_aOTC was neither close to the CP binding site nor connected to the trimer center. In addition, results from the thermal shift assay indicate that each OTC prefers the substrate for the unidirectional process. The active site exhibited a blocked binding site for CP in the Ps_cOTC structure, whereas residues at the active site in Ps_aOTC established a binding site to facilitate CP binding. Our data provide novel insights into the unidirectional catalysis of OTCs and cooperativity, which are distinguishable features of two metabolically specialized proteins.

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4′,6-dimethoxyisoflavone-7-O-β-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1β and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.

Abstract Background S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. Results A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. Conclusions Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure–function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.

Total fatty-acid (FA) contents of different organs (stomach, liver, brain, and skin) of two Antarctic fish, marbled rockcod (Notothenia rossii) and mackerel icefish (Champsocephalus gunnari), were examined using gas chromatography–mass spectrometry (GC–MS). N. rossii possessed higher contents of total omega-3, where eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the most represented omega-3 FAs, were distributed throughout all parts of the fish. The highest level of EPA was observed in the skin and that of DHA was observed in the brain of N. rossii. C. gunnari showed organ peculiarity in that most of the omega-3 FAs were found in stomach and skin. Specifically, the highest levels of EPA and DHA were both observed in the stomach. Although N. rossii and C. gunnari both inhabit the Antarctic Southern Oceans, their characteristics in terms of the composition of fatty acids were shown to vary. The extracts were also evaluated for matrix metalloproteinase-1 (MMP-1)-inhibitory activities in UVB-induced human dermal fibroblasts, where extracts of the skin and liver of N. rossii showed the most significant inhibition upon MMP-1 production. These findings provide experimental evidence that the extracts of the Antarctic fish could be utilized as bioactive nutrients, particularly in the enhancement of skin health.

The stress-responsive, SK5 subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments.

Three p-terphenyls (2–4)—2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)—were isolated for the first time as natural products along with seven known compounds (1, 5–10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 μM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 μM.

Single-stranded DNA-binding proteins (SSBs) are essential for DNA metabolism, including repair and replication, in all organisms. SSBs have potential applications in molecular biology and in analytical methods. In this study, for the first time, we purified, structurally characterized, and analyzed psychrophilic SSB (LjSSB) from Lacinutrix jangbogonensis PAMC 27137 isolated from the Antarctic region. LjSSB has a relatively short amino acid sequence, consisting of 111 residues, with a molecular mass of 12.6 kDa. LjSSB protein was overexpressed in Escherichia coli BL21 (DE3) and analyzed for binding affinity using 20- and 35-mer deoxythymidine oligonucleotides (dT). In addition, the crystal structure of LjSSB at a resolution 2.6 Å was obtained. The LjSSB protein crystal belongs to the space group C222 with the unit cell parameters of a = 106.58 Å, b = 234.14 Å, c = 66.14 Å. The crystal structure was solved using molecular replacement, and subsequent iterative structure refinements and model building are currently under progress. Further, the complete structural information of LjSSB will provide a novel strategy for protein engineering and for the application on molecular biological techniques.

Ester prodrugs can develop novel antibiotics and have potential therapeutic applications against multiple drug-resistant bacteria. The antimicrobial activity of these prodrugs is activated after being cleaved by the esterases produced by the pathogen. Here, novel esterase ScEst originating from Staphylococcus chromogenes NCTC10530, which causes dairy cow mastitis, was identified, characterized, and analyzed using X-ray crystallography. The gene encoding ScEst was cloned into the pVFT1S vector and overexpressed in E. coli. The recombinant ScEst protein was obtained by affinity and size-exclusion purification. ScEst showed substrate preference for the short chain length of acyl derivatives. It was crystallized in an optimized solution composed of 0.25 M ammonium citrate tribasic (pH 7.0) and 20% PEG 3350 at 296 K. A total of 360 X-ray diffraction images were collected at a 1.66 Å resolution. ScEst crystal belongs to the space group of P212121 with the unit cell parameters of a = 50.23 Å, b = 68.69 Å, c = 71.15 Å, and α = β = γ = 90°. Structure refinement after molecular replacement is under progress. Further biochemical studies will elucidate the hydrolysis mechanism of ScEst. Overall, this study is the first to report the functional characterization of an esterase from Staphylococcus chromogenes, which is potentially useful in elaborating its hydrolysis mechanism.

Stereocalpin B, a new cyclic depsipeptide (1), and a new dibenzofuran derivative (3), were isolated from the Antarctic lichen, Ramalina terebrata (Ramalinaceae), along with a known cyclic depsipeptide (2). The structures of new compounds were characterized by comprehensive spectrometric analyses; high-resolution fast atom bombardment mass spectrometry (HR-FABMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Stereocalpin B (1) existed in a rotameric equilibrium, which was confirmed using nuclear Overhauser effect spectroscopy (NOESY)/exchange spectroscopy (EXSY) spectrum. Absolute configurations of the amino acid units in 1 were assigned using the advanced Marfey’s method and subsequent NOESY analysis of the 5-hydroxy-2,4-dimethyl-3-oxo-decanoic acid residue confirmed the complete stereochemistry of 1. Compounds 1-3 exhibited moderate antimicrobial activities against E. coli, with the IC50 values ranging from 18–30 μg/mL. Compound 2 exhibited cell growth inhibition against HCT116 cell lines, with the IC50 value of 20 ± 1.20 μM, and compounds 1 and 2 also showed potent anti-inflammatory activities against lipopolysaccharide (LPS)-induced RAW264.7 macrophages with the IC50 values ranging from 5–7 μM.

A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.

Two undescribed rearranged cadinane-type sesquiterpenoids (1–2), named sinulaketol A-B, together with one new chlorinated steroid (3), one new gorgosterol (4), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7–8) were isolated from the soft coral Sinularia brassica. The structures of these compounds were established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. Their absolute configurations were also determined by the ECD calculations and DP4+ probability analysis. Antileishmanial activity of compounds 1–8 was evaluated in vitro against the amastigote forms of Leishmania donovani, in which compounds 3, 6, and 7 inhibited the growth of L. donovani by 58.7, 74.3, 54.7%, respectively, at a concentration of 50 μM. Antimicrobial effect of the isolated compounds were also evaluated against Candida albicans, Staphylococcus aureus, and Escherichia coli. Compound 6, a brominated diterpene, exhibited antimicrobial effect against S. aureus.

This report deals with the purification, characterization, and a preliminary crystallographic study of a novel cold-active esterase (HaEst1) from Halocynthiibacter arcticus. Primary sequence analysis reveals that HaEst1 has a catalytic serine in G-x-S-x-G motif. The recombinant HaEst1 was cloned, expressed, and purified. SDS-PAGE and zymographic analysis were carried out to characterize the properties of HaEst1. A single crystal of HaEst1 was obtained in a solution containing 10% (w/v) PEG 8000/8% ethylene glycol, 0.1 M Hepes-NaOH, pH 7.5. Diffraction data were collected to 2.10 Å resolution with P21 space group. The final Rmerge and Rp.i.m values were 7.6% and 3.5% for 50–2.10 Å resolution. The unit cell parameters were a = 35.69 Å, b = 91.21 Å, c = 79.15 Å, and β = 96.9°.

Phytochemicals are known to have anti-inflammatory effects in vitro and in vivo, such as in inflammatory disease model systems. Inflammation is an essential immune response to exogenous stimuli such as infection and injury. Although inflammation is a necessary host-defense mechanism, chronic inflammation is associated with the continuous local or systemic release of inflammatory mediators, non-cytokine mediators, such as ROS and NO, and inflammatory cytokines are strongly implicated in the pathogenesis of various inflammatory disorders. Phytochemicals that exhibit anti-inflammatory mechanisms that reduce sustained inflammation could be therapeutic candidates for various inflammatory diseases. These phytochemicals act by modulating several main inflammatory signaling pathways, including NF-κB, MAPKs, STAT, and Nrf-2 signaling. Here, we discuss the characteristics of phytochemicals that possess anti-inflammatory activities in various chronic inflammatory diseases and review the molecular signaling pathways altered by these anti-inflammatory phytochemicals, with a focus on transcription factor pathways. Furthermore, to evaluate the phytochemicals as drug candidates, we translate the effective doses of phytochemicals in mice or rat disease models into the human-relevant equivalent and compare the human-relevant equivalent doses of several phytochemicals with current anti-inflammatory drugs doses used in different types of chronic inflammatory diseases.

Ice-binding proteins (IBPs) have ice recrystallization inhibition (IRI) activity. IRI property has been extensively utilized for the cryopreservation of different types of cells and tissues. Recent reports demonstrated that IRI can also play a significant role in protecting proteins from freezing damage during freeze–thaw cycles. In this study, we hypothesized that the protective capability of IBPs on proteins against freeze–thaw damage is proportional to their IRI activity. Hence we used two IBPs: one with higher IRI activity (LeIBP) and the other with lower activity (FfIBP). Yeast alcohol dehydrogenase (ADH) was used as a freeze-labile model protein. IBPs and ADH were mixed, frozen at −20 °C, and thawed repeatedly. The structure of ADH was assessed using fluorescence emission spectra probed by 1-anilinonaphthalene-8-sulfonate over the repeated freeze–thaw cycles. The activity was monitored at 340 nm spectrophotometrically. Fluorescence data and activity clearly indicated that ADH without IBP was freeze-labile. However, ADH maintained about 70% residual activity after five repeated cycles at a minimal concentration of 0.1 mg mL-1 of high IRI-active LeIBP, but only 50% activity at 4 mg mL−1 of low active FfIBP. These results showed that the protection of proteins from freeze–thaw stress by IBPs is proportional to their IRI activity.

Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology.

Hormone sensitive lipase is a central enzyme in triacylglycerol hydrolysis, lipid modification, and transformation of various lipids. Microbial hormone-sensitive lipases, which are highly similar to a catalytic domain of mammalian equivalents, have attracted strong attention due to their application potentials. Here, characterization and a preliminary X-ray crystallographic analysis of a novel bacterial homologue of hormone-sensitive lipase (HaLip1) from Halocynthiibacter arcticus is reported. Sequence analysis shows that HaLip1 has a conserved serine residue within the GDSAG motif. In addition, a characteristic HGGG motif for oxyanion formation was identified. The HaLip1 protein was overexpressed in E. coli. SDS-PAGE, overlay assay, and mass analysis were performed to confirm purity and activity of HaLip1 protein. Furthermore, HaLip1 was crystallized in a condtion consisting of 25% (w/v) PEG 3350, 0.1 M Hepes-KOH, pH 7.5, 0.2 M sodium chloride. Diffraction data were processed to 1.30 Å with an Rmerge of 7.3%. The crystals of HaLip1 belong to the P212121, with unit cell parameters of a = 54.6 Å, b = 59.5 Å, and c = 82.9 Å.

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4°C than at 25°C. We solved the crystal structure of PbAcE at 2.1-Å resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the β4-α3 and β5-α4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

The Arctic moss Aulacomnium turgidum (Wahlenb.) Schwaegr. is distributed widely above the Arctic Circle and can regenerate successfully after 400 years of ice entombment. Here, we report the complete mitogenome sequence of A. turgidum (103,937 bp). The genome contains 3 ribosomal RNAs, 24 transfer RNAs, and 40 protein-encoding genes. In a phylogenetic tree generated using the combined amino acid sequences of 32 mitochondrial genes from A. turgidum, 25 Bryophyta, and three Marchantiophyta, the phylogenetic position of A. turgidum (Rhizogoniales) is close to that of the Hypnales and Ptychomniales, forming a monophyletic clade with perfect supporting values.