Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at ?12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.     

Cloning and characterization of a novel cold-active endoglucanase establishing a new subfamily of glycosyl hydrolase family 5 from a psychrophilic deep-sea bacterium

The gene of a novel endo-β-1,4-glucanase (named Cel5M) was isolated from the psychrophilic deep-sea bacteria Pseudomonas sp. MM15. The deduced protein sequence lacked the typical cellulase domain structures of the carbohydrate-binding module and the linker region. Cel5M showed relatively higher activity toward carboxymethyl cellulose, but much lower activity toward p-nitrophenyl-β-D-galactopyranoside and no activity toward avicel. Cel5M was identified as a cold-active cellulase with an optimal temperature of 30 °C and it was active within a narrow pH range with an optimum at pH 4.5. Phylogenetic analysis showed that Cel5M represented a new subfamily of the glycosyl hydrolase family 5, representing an opportunity for research into and applications of novel cold-active cellulases.     

Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida

AIMS: This study was conducted to determine optimal conditions for pectate lyase (PL) production by two psychrophilic yeast strains and to compare the properties of the cold-active enzymes using mesophilic PL as reference enzyme. METHODS AND RESULTS: Two psychrophilic yeasts isolated from remote geographical locations (European Alps, north Siberia) produced extracellular cold-active PL. Both strains were identified as Mrakia frigida by analysis of ITS and large subunit (LSU) rRNA sequences. Maximum enzyme production occurred at a cultivation temperature of 1 or 5 degrees C. The apparent optimum for enzyme activity was observed at 30 degrees C and pH 8.5-9. The enzymes were thermolabile, but were resistant to repeated freezing and thawing. CONCLUSION: We describe for the first time alkaline PL-producing representatives of the yeast species M. frigida. The two strains produce cold-active PL with similar properties, but have a different enzyme production pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzymes described in this study could be useful for a wide range of applications, such as low-temperature pretreatment of wastewater containing pectic substances.     

Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a β-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.     

Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast

Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993) Nature 361, 736-739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min(-1)) by a factor of 5 x 10(6) and that they have a very low affinity for its preferred substrate (K(m) = 592 micrometer). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.     

Cloning,expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda

A new xylanase gene (xynA) from the marine microorganism Zunongwangia profunda was identified to encode 374 amino acid residues. Its product (XynA) showed the highest identity (42.78 %) with a xylanase from Bacillus sp. SN5 among the characterized xylanases. XynA exhibited the highest activity at pH 6.5 and 30 °C, retaining 23 and 38 % of the optimal activity at 0 and 5 °C, respectively. XynA was not only cold active, but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (180 % of the activity) at 3 M NaCl and retaining nearly 100 % activity at 5 M NaCl, compared to the absence of NaCl. In the presence of 3 M NaCl, the k _cat/K _m value of XynA exhibited a 3.41-fold increase for beechwood xylan compared to no added NaCl, and the residual activity of XynA increased from 23 % (no added NaCl) to 58 % after 1 h incubation at 45 °C. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from 3 to 5 M. The features of cold activity and salt tolerance suggest the potential application of XynA in the food industry and bioethanol production from marine seaweeds.     

Biochemical and biophysical characterization of a novel plant protein disulfide isomerase

We recently isolated a protein disulfide isomerase (PDI) from the Rubiaceae (coffee family) plant Oldenlandia affinis (OaPDI) and demonstrated that it facilitates the production of disulfide-knotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including size-exclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers and/or higher-order oligomers were observed at increased conditions, raising the possibility that dimerization and/or oligomerization could be a mechanism to adapt to the various-sized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI.     

Xylanase from the psychrophilic yeast Cryptococcus adeliae

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 ± 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0°–20°C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices. Received: April 20, 1999 / Accepted: January 13, 2000     

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).     

Kazachstania psychrophila sp. nov., a novel psychrophilic yeast isolated from vacuum-packed beef

Five novel ascosporogenous yeast strains (H382, H396, H409, H433^T and H441) were found through a survey of vacuum-packed beef microbiota. Sequence analysis of ITS domain and LSU rRNA genes showed that the new strains represent a distinct lineage within the genus Kazachstania, closely related to Kazachstania lodderae (97.0 % identity) and Kazachstania ichnusensis (96.1 % identity). The main difference of strains H382, H396, H409, H433^T and H441 to strains of known Kazachstania species is the maximum growth temperature, which is below 20 °C for the new strains, whereas related species grow at 25 °C. Furthermore, the strains differed from known Kazachstania species in assimilation and fermentation patterns of carbon sources. Based on these characteristics, the five strains are considered to represent a novel species of the genus Kazachstania for which the name Kazachstania psychrophila sp. nov. is proposed. The type strain is H433^T (DSM 26230^T=CBS 12689^T). The Mycobank number of the type strain is MB 803980.     

Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, <Emphasis Type="Italic">Glaciozyma antarctica</Emphasis>

The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.     

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.     

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.