scholarly journals Identification and enzymatic analysis of an archaeal ATP-dependent serine kinase from the hyperthermophilic archaeon Staphylothermus marinus

2021 ◽  
Author(s):  
Yasunobu Mori ◽  
Hiroki Kawamura ◽  
Takaaki Sato ◽  
Takayuki Fujita ◽  
Ryuhei Nagata ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Sugar Metabolism ◽  
Inorganic Polyphosphates ◽  
Serine Kinase ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Wide Range ◽  
Genes Encoding ◽  
Phosphate Donor ◽  
Domains Of Life

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis ( Tk -SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk -SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk -SerK from Euryarchaeota (42-45% identical). Tk - serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli . All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity towards l -Ser, but not with other compounds including d -Ser, l -threonine and l -homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as the phosphate donor. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5’-triphosphates compared to triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis ( Tk -SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk -SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

scholarly journals Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Takahiro Shimosaka ◽  
Kira S. Makarova ◽  
Eugene V. Koonin ◽  
Haruyuki Atomi
Keyword(s):  
Metabolic Pathways ◽  
Coenzyme A ◽  
Protein A ◽  
Biosynthesis Pathway ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Uncharacterized Gene ◽  
Wide Range ◽  
Insight Into

ABSTRACT Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon Thermococcus kodakarensis encodes two homologs of bacterial DPCK and the DPCK domain of eukaryotic CoA synthase, TK1334 and TK2192. We purified the recombinant TK1334 and TK2192 proteins and found that they lacked DPCK activity. Bioinformatic analyses showed that, in several archaea, the uncharacterized gene from arCOG04076 protein is fused with the gene for phosphopantetheine adenylyltransferase (PPAT), which catalyzes the reaction upstream of the DPCK reaction in CoA biosynthesis. This observation suggested that members of arCOG04076, both fused to PPAT and standalone, could be the missing archaeal DPCKs. We purified the recombinant TK1697 protein, a standalone member of arCOG04076 from T. kodakarensis, and demonstrated its GTP-dependent DPCK activity. Disruption of the TK1697 resulted in CoA auxotrophy, indicating that TK1697 encodes a DPCK that contributes to CoA biosynthesis in T. kodakarensis. TK1697 homologs are widely distributed in archaea, suggesting that the arCOG04076 protein represents a novel family of DPCK that is not homologous to bacterial and eukaryotic DPCKs but is distantly related to bacterial and eukaryotic thiamine pyrophosphokinases. We also constructed and characterized gene disruption strains of TK0517 and TK2128, homologs of bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and PPAT, respectively. Both strains displayed CoA auxotrophy, indicating their contribution to CoA biosynthesis. Taken together with previous studies, the results experimentally validate the entire CoA biosynthesis pathway in T. kodakarensis. IMPORTANCE CoA is utilized in a wide range of metabolic pathways, and its biosynthesis is essential for all life. Pathways for CoA biosynthesis in bacteria and eukaryotes have been established. In archaea, however, the enzyme that catalyzes the final step in CoA biosynthesis, dephospho-CoA kinase (DPCK), had not been identified. In the present study, bioinformatic analyses identified a candidate for the DPCK in archaea, which was biochemically and genetically confirmed in the hyperthermophilic archaeon Thermococcus kodakarensis. Genetic analyses on genes presumed to encode bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and phosphopantetheine adenylyltransferase confirmed their involvement in CoA biosynthesis. Taken together with previous studies, the results reveal the entire pathway for CoA biosynthesis in a single archaeon and provide insight into the different mechanisms of CoA biosynthesis and their distribution in nature.

scholarly journals TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis

2021 ◽  
Author(s):  
Ren-Chao Zheng ◽  
Xia-Feng Lu ◽  
Hiroya Tomita ◽  
Shin-ichi Hachisuka ◽  
Yu-Guo Zheng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Surface Glycoprotein ◽  
Lag Phase ◽  
Class Iii ◽  
Cell Surface Glycoprotein ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Domains Of Life ◽  
Amino Acid Racemase

Members of Thermococcales harbor a number of genes encoding putative aminotransferase Class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell-surface glycoprotein gene TK0895 (Pcsg). The purified protein did not display aminotransferase activity, but exhibited racemase activity. An examination on most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal media supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase, but displayed cell yield similar to that observed in medium with l-Leu. By contrast, the ΔTK1211 strain displayed growth in medium with l-Leu, but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells, and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. IMPORTANCE Phylogenetic analysis of aminotransferase Class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups can now be presumed to function as a broad substrate specificity amino acid racemase (Subgroup 1), alanine/serine racemase (Subgroup 2), ornithine ω-aminotransferase (Subgroup 3), or Leu/Met racemase (Subgroup 4).

scholarly journals Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

2005 ◽  
Vol 187 (20) ◽  
pp. 7038-7044 ◽  
Author(s):  
Takeshi Tanaka ◽  
Fumikazu Takahashi ◽  
Toshiaki Fukui ◽  
Shinsuke Fujiwara ◽  
Haruyuki Atomi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Abc Transporter ◽  
Catalytic Properties ◽  
Sugar Metabolism ◽  
Amino Sugar ◽  
Hyperthermophilic Archaeon ◽  
Chitin Degradation ◽  
Genes Encoding ◽  
Activity Dependent

ABSTRACT A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD Tk ) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD Tk was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD Tk clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD Tk . In T. kodakaraensis cells, glmDTk was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-β-glucosaminidase gene (glmATk ) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD Tk is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

Endonuclease V from the archaeon Thermococcus kodakarensis is an inosine-specific ribonuclease

2021 ◽  
Author(s):  
Miyako Shiraishi ◽  
Michihi Hidaka ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Nucleic Acid ◽  
Potential Role ◽  
Biochemical Properties ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Substrate Preferences ◽  
Endonuclease V ◽  
Domains Of Life

Abstract Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA which is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in regulation of inosine-containing RNA.

scholarly journals A Phosphofructokinase Homolog fromPyrobaculum calidifontisDisplays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Iram Aziz ◽  
Tahira Bibi ◽  
Naeem Rashid ◽  
Riku Aono ◽  
Haruyuki Atomi ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Catalytic Efficiency ◽  
Content Type ◽  
Reading Frame ◽  
Thermococcus Kodakarensis ◽  
Pyrobaculum Calidifontis ◽  
Cell Extracts ◽  
Activity Measurements ◽  
Phosphate Donor ◽  
Sugar Phosphates

ABSTRACTThe genome of the hyperthermophilic archaeonPyrobaculum calidifontiscontains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) fromAeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase fromDesulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1· s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts ofP. calidifontisindicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) inThermococcus kodakarensisthat functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCEThe discovery of the pentose bisphosphate pathway inThermococcus kodakarensishas clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase inPyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

scholarly journals The Fennoscandian Shield deep terrestrial virosphere suggests slow motion ‘boom and burst’ cycles

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Karin Holmfeldt ◽  
Emelie Nilsson ◽  
Domenico Simone ◽  
Margarita Lopez-Fernandez ◽  
Xiaofen Wu ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Nutrient Recycling ◽  
Slow Motion ◽  
Deep Biosphere ◽  
Wide Range ◽  
Domains Of Life ◽  
Microbial Turnover ◽  
Rna Transcript ◽  
Viral Isolates ◽  
Antagonistic Interactions

AbstractThe deep biosphere contains members from all three domains of life along with viruses. Here we investigate the deep terrestrial virosphere by sequencing community nucleic acids from three groundwaters of contrasting chemistries, origins, and ages. These viromes constitute a highly unique community compared to other environmental viromes and sequenced viral isolates. Viral host prediction suggests that many of the viruses are associated with Firmicutes and Patescibacteria, a superphylum lacking previously described active viruses. RNA transcript-based activity implies viral predation in the shallower marine water-fed groundwater, while the deeper and more oligotrophic waters appear to be in ‘metabolic standby’. Viral encoded antibiotic production and resistance systems suggest competition and antagonistic interactions. The data demonstrate a viral community with a wide range of predicted hosts that mediates nutrient recycling to support a higher microbial turnover than previously anticipated. This suggests the presence of ‘kill-the-winner’ oscillations creating slow motion ‘boom and burst’ cycles.

scholarly journals A Transcriptomic Approach to the Metabolism of Tetrapyrrolic Photosensitizers in a Marine Annelid

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3924
Author(s):  
Maria Leonor Santos ◽  
Mariaelena D’Ambrosio ◽  
Ana P. Rodrigo ◽  
A. Jorge Parola ◽  
Pedro M. Costa
Keyword(s):  
Metabolic Pathways ◽  
Molecular Networks ◽  
Bile Pigments ◽  
Rna Seq ◽  
The Past ◽  
Wide Range ◽  
Genes Encoding ◽  
Organ Specific ◽  
Bright Green ◽  
Green Coloration

The past decade has seen growing interest in marine natural pigments for biotechnological applications. One of the most abundant classes of biological pigments is the tetrapyrroles, which are prized targets due their photodynamic properties; porphyrins are the best known examples of this group. Many animal porphyrinoids and other tetrapyrroles are produced through heme metabolic pathways, the best known of which are the bile pigments biliverdin and bilirubin. Eulalia is a marine Polychaeta characterized by its bright green coloration resulting from a remarkably wide range of greenish and yellowish tetrapyrroles, some of which have promising photodynamic properties. The present study combined metabolomics based on HPLC-DAD with RNA-seq transcriptomics to investigate the molecular pathways of porphyrinoid metabolism by comparing the worm’s proboscis and epidermis, which display distinct pigmentation patterns. The results showed that pigments are endogenous and seemingly heme-derived. The worm possesses homologs in both organs for genes encoding enzymes involved in heme metabolism such as ALAD, FECH, UROS, and PPOX. However, the findings also indicate that variants of the canonical enzymes of the heme biosynthesis pathway can be species- and organ-specific. These differences between molecular networks contribute to explain not only the differential pigmentation patterns between organs, but also the worm’s variety of novel endogenous tetrapyrrolic compounds.

scholarly journals Newly discovered Asgard archaea Hermodarchaeota potentially degrade alkanes and aromatics via alkyl/benzyl-succinate synthase and benzoyl-CoA pathway

2021 ◽  
Author(s):  
Jia-Wei Zhang ◽  
Hong-Po Dong ◽  
Li-Jun Hou ◽  
Yang Liu ◽  
Ya-Fei Ou ◽  
...  
Keyword(s):  
Nitrate Reduction ◽  
Coenzyme A ◽  
Organic Substrates ◽  
Mangrove Sediments ◽  
Sulfate Reducing ◽  
Sequence Read Archive ◽  
Wide Range ◽  
Genes Encoding ◽  
Sediment Carbon ◽  
Reducing Bacteria

AbstractAsgard archaea are widely distributed in anaerobic environments. Previous studies revealed the potential capability of Asgard archaea to utilize various organic substrates including proteins, carbohydrates, fatty acids, amino acids and hydrocarbons, suggesting that Asgard archaea play an important role in sediment carbon cycling. Here, we describe a previously unrecognized archaeal phylum, Hermodarchaeota, affiliated with the Asgard superphylum. The genomes of these archaea were recovered from metagenomes generated from mangrove sediments, and were found to encode alkyl/benzyl-succinate synthases and their activating enzymes that are similar to those identified in alkane-degrading sulfate-reducing bacteria. Hermodarchaeota also encode enzymes potentially involved in alkyl-coenzyme A and benzoyl-coenzyme A oxidation, the Wood–Ljungdahl pathway and nitrate reduction. These results indicate that members of this phylum have the potential to strictly anaerobically degrade alkanes and aromatic compounds, coupling the reduction of nitrate. By screening Sequence Read Archive, additional genes encoding 16S rRNA and alkyl/benzyl-succinate synthases analogous to those in Hermodarchaeota were identified in metagenomic datasets from a wide range of marine and freshwater sediments. These findings suggest that Asgard archaea capable of degrading alkanes and aromatics via formation of alkyl/benzyl-substituted succinates are ubiquitous in sediments.

scholarly journals Butyrivibrio hungateiMB2003 Competes Effectively for Soluble Sugars Released byButyrivibrio proteoclasticusB316Tduring Growth on Xylan or Pectin

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Nikola Palevich ◽  
William J. Kelly ◽  
Siva Ganesh ◽  
Jasna Rakonjac ◽  
Graeme T. Attwood
Keyword(s):  
Bacterial Species ◽  
Soluble Sugars ◽  
Plant Fiber ◽  
Content Type ◽  
Plant Polysaccharide ◽  
Rumen Microbes ◽  
Carbohydrate Degradation ◽  
Wide Range ◽  
Genes Encoding ◽  
Enzymatic Machinery

ABSTRACTRumen bacterial species belonging to the genusButyrivibrioare important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question,Butyrivibrio hungateiMB2003 andButyrivibrio proteoclasticusB316Twere grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316Twas able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316Tgrown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316Twhen either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316Tand MB2003 was assessed; B316Ttranscription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316Tfor the released sugars. These results suggest that B316Thas a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCEFeeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation.Butyrivibriospecies are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species ofButyrivibriohave developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber.

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