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.
Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.
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.
Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 A. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.
Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15 °C, 0 °C and -12 °C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12 °C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12 °C compared to 0 °C and 15 °C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.
Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.
Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.
Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.
The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.
Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.