An evidence of laccases in archaea

Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution, e.g. Haloarcula marismortui ATCC 43049, Natronomonas pharaonis DSM2160, Pyrobaculum aerophilum IM2, Candidatus Nitrosopumilus maritimus SCM1, Halorubrum lacusprofundi ATCC 49239. Most of the archaeal multicopper oxidases reported here are of Type 1 and Type 2 whereas type 3 copper-binding domain could be found in Pyrobaculum aerophilum IM2 and Halorubrum lacusprofundi ATCC49239. An analysis of the genome sequence database revealed the presence of novel types of two-domain laccases in archaea. ed using this method. CyMVin the positive samples of Phalaenopsis sp. and Arachnis sp. was confirmed by DNA sequencing and cp gene homeology blast. The results showed that CyMV extracted from the leaves of orchid in Hangzhou, Zhejiang Province, China, could be derived from Kunming city (KM), Yunnan Province, China. This method characterized by high sensitivity, specificity, and precision is suitable for early diagnosis and quantitative detection of CyMV.     

Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles in Sulfolobus sp. emerge from a specific budding process with similarity to the endosomal sorting pathway.     

Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus

In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 μM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

中国和美国原始土壤中非高温泉古菌的发现和鉴定

近年来在非极端环境中已经发现有古菌(Archaea)的存在, 但在中国原始土壤中还未见报道。本研究的目的是调查古菌是否存在于两个分别取自中国新疆和广西的土壤及两个美国亚利桑那州南部地区的土壤中。我们分别构建了这四个原始土壤的古菌16S rDNA文库并对28个克隆的16S rDNA进行了鉴定。所有这些16S rDNA的序列都归类于古菌的泉古菌门(Crenarchaeota)。进化树分析表明, 这些泉古菌的16S rDNA属于非高温陆地环境中的泉古菌种群, 明显区别于海洋和淡水地带的泉古菌种群。这个泉古菌种群又有两个分支, 这两个分支在16S rDNA序列上和G C含量上有明显的区别。本研究在两个中国和两个美国原始土壤中鉴定了非高温泉古菌的存在, 由此证明泉古菌的存在范围不只局限于高温等极端环境。另外, 美国原始土壤中的泉古菌只属于一个进化分支, 这说明非高温泉古菌种群的类型和土壤的地理位置及土壤特性有关。     

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment.     

A review of acquired thermotolerance, heat-shock proteins, and molecular chaperones in archaea

Abstract: Acquired thermotolerance, the associated synthesis of heat-shock proteins (HSPs) under stress conditions, and the role of HSPs as molecular chaperones under normal growth conditions have been studied extensively in eukaryotes and bacteria, whereas research in these areas in archaea is only beginning. All organisms have evolved a variety of strategies for coping with high-temperature stress, and among these strategies is the increased synthesis of HSPs. The facts that both high temperatures and chemical stresses induce the HSPs and that some of the HSPs recognize and bind to unfolded proteins in vitro have led to the theory that the function of HSPs is to prevent protein aggregation in vivo. The facts that some HSPs are abundant under normal growth conditions and that they assist in protein folding in vitro have led to the theory that they assist protein folding in vivo; in this role, they are referred to as molecular chaperones. The limited research on acquired thermotolerance, HSPs, and molecular chaperones in archaea, particularly the hyperthermophilic archaea, suggests that these extremophiles provide a new perspective in these areas of research, both because they are members of a separate phylogenetic domain and because they have evolved to live under extreme conditions.     

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.     

Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.