Hak Jun Kim

An extracellular ice-binding glycoprotein from an Arctic psychrophilic yeast.

A psychrophilic yeast was isolated from an Arctic pond and its culture supernatant showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of approximately 26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yeast IBP was determined and was found to encode a 261 amino acid protein with molecular weight of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria, clustering with a class of ice-active proteins.

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast

Background: Ice-binding proteins improve the cold tolerance of cells by inhibiting ice growth and recrystallization. Results: Crystal structure and mutagenesis data of LeIBP suggests the B face as an ice-binding site. Conclusion: LeIBP structure adopts a β-helical fold and the aligned Thr/Ser/Ala residues are critical for ice binding. Significance: LeIBP structure can serve as a structural model for a large number of IBPs. Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

Possible Role of Horizontal Gene Transfer in the Colonization of Sea Ice by Algae

Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Proteins with similar sequences and ice-binding activities are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 47% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes.

Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica

Abstract Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica.

Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state.

The shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor‐liganded and tetrahedral intermediate (TI)‐liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI‐liganded structure provides absolute configuration of the C‐2 atom from the F‐PEP moiety of the enzyme‐bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate‐bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new‐generation antibiotics.

Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30☆

Previously, we reported the ice-binding protein (LeIBP) from the Arctic yeast Leucosporidium sp. AY30. In this study we provide physicochemical characterization of this IBP, which belongs to a class of IBPs that exhibited no significant similarity in primary structure to other known antifreeze proteins (AFPs). We compared native, glycosylated and non-glycosylated recombinant LeIBPs. Interestingly, size-exclusion chromatography and analytical ultracentrifugation revealed that LeIBP self-associates with a reversible dimer with K(d) values in the range 3.45-7.24×10(-6) M. Circular dichroism (CD) spectra showed that LeIBP, glycosylated or non-glycosylated, is predominantly composed of β-strand secondary structural elements (54.6%), similar to other β-helical antifreeze proteins (AFPs). In thermal hysteresis (TH) activity measurements, native LeIBP was twice more active (0.87 °C at 15 mg/mL) than that of the recombinant IBPs (0.43-0.42 °C at 10.8 mg/mL). This discrepancy is probably due to uncharacterized enhancing factors carried over during ice affinity purification, because glycosylated and non-glycosylated recombinant proteins displayed similarly low activity. Ice recrystallization inhibition (RI) activities of the native and recombinant LeIBPs were comparable. Measurements of CD, TH activity, and RI showed that glycosylation does not cause structural changes and is not required for function. An ice-etching experiment using green fluorescent protein-tagged IBP revealed that LeIBP binds, just as hyperactive AFPs, to both basal and pyramidal prism planes of the ice crystal. Taken together, our results indicate that LeIBP, structurally similar to hyperactive AFPs, is moderately active and that a reversible dimer has no effect on its activity.

Pseudomonas pelagia sp. nov., isolated from a culture of the Antarctic green alga Pyramimonas gelidicola.

Two Gram-negative, strictly aerobic bacterial strains, designated CL-AP6(T) and CL-AP22, were isolated from a culture of a green alga, Pyramimonas gelidicola, established from the Antarctic. Cells of the strains were straight rods and motile by means of a single polar flagellum. The strains grew with 0.5-8 % (w/v) NaCl (optimum, 1-2 %) and at 4-33 degrees C (optimum, 25 degrees C) and pH 6.5-9.1 (optimum, pH 7.5-8.1). The two strains shared 98.8 % 16S rRNA gene sequence similarity. Analysis of the 16S rRNA gene sequences of strains CL-AP6(T) and CL-AP22 revealed that they were members of the genus Pseudomonas and were most closely related to Pseudomonas xiamenensis C10-2(T) (95.5-95.8 % sequence similarity) and next to Pseudomonas pertucinogena NBRC 14163(T) (95.1-95.5 % sequence similarity) and to other members of the genus Pseudomonas (<95.2 % sequence similarity). Phylogenetic analyses based on the 16S rRNA gene sequences showed that strain CL-AP6(T) formed a robust clade with strain CL-AP22, and that this clade clustered tightly with the nearest clade containing P. xiamenensis and P. pertucinogena. The major isoprenoid quinone of strains CL-AP6(T) and CL-AP22 was Q-9 and the major cellular fatty acids were C(18 : 1)omega7c (40.2-41.6 %), summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 26.3-26.8 %), C(16 : 0) (13.7-13.9 %) and C(12 : 0) (5.8-6.2 %). The genomic DNA G+C contents of strains CL-AP6(T) and CL-AP22 were 59.1 and 57.2 mol%, respectively. DNA-DNA hybridization experiments revealed high relatedness values (98.5+/-0.5 %; mean+/-sem, n=2) between strains CL-AP6(T) and CL-AP22, indicating that the two strains constituted a single species. However, the two strains differed phenotypically from P. xiamenensis by their inability to grow without NaCl, temperature range for growth, hydrolysis of starch and production of certain enzymes. The phylogenetic analysis and physiological and biochemical data showed that strains CL-AP6(T) and CL-AP22 should be classified as representing a novel species in the genus Pseudomonas, for which the name Pseudomonas pelagia sp. nov. is proposed. The type strain is CL-AP6(T) (=KCCM 90073(T)=JCM 15562(T)).

Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr)n with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs.

Moritella dasanensis sp. nov., a psychrophilic bacterium isolated from the Arctic ocean

An aerobic, motile, Gram-negative, ice-active substance-producing, rod-shaped psychrophile, designated strain ArB 0140T, was isolated from seawater collected from near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain ArB 0140T showed a distinct phyletic line within the genus Moritella. Characteristic chemotaxonomic data [predominant isoprenoid quinone, Q8; major fatty acids, C14 : 0, C14 : 1, C16 : 0, C16 : 1 and C22 : 6 (docosahexaenoic acid; DHA)] also corroborated the affiliation of strain ArB 0140T to the genus Moritella. The maximal growth rate of the novel strain was observed at 9 degrees C, with a maximum temperature for growth of 18 degrees C. The genomic DNA G+C content was 46.9 mol%. Based on the data obtained from this polyphasic study, including DNA-DNA relatedness, physiological and biochemical tests and ice-controlling activity, strain ArB 0140T was found to be genetically and phenotypically different from other recognized species of the genus Moritella. Therefore strain ArB 0140T represents a novel species, for which the name Moritella dasanensis sp. nov. is proposed. The type strain is ArB 0140T (=KCTC 10814T=KCCM 42845T=JCM 14759T).

Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H

The putative lipase CpsLip from the psychrophilic bacterium Colwellia psychrerythraea 34H encodes a 34,538 Da, 308-amino-acid protein. In this study, CpsLip (UniProtKB code Q486T5) was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli and purified by affinity and size-exclusion chromatography. The expression and purification of CpsLip enabled characterization of the lipase enzymatic properties of the protein. The optimal activity temperature and pH of the recombinant protein were 298 K and pH 7, respectively. CpsLip maintained over 80% activity in the low-temperature range (278-288 K), thereby suggesting that CpsLip is a cold-active lipase. Substrate-specificity analysis demonstrated that CpsLip exhibits maximum activity towards the C12 acyl group. In addition, sequence-alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135 and His283. Moreover, purified CpsLip was successfully crystallized using the hanging-drop vapour-diffusion method and a complete diffraction data set was collected to 4.0 Å resolution using synchrotron radiation on the BL-5A beamline of the Photon Factory.