Hyperthermophilic archaea as a source for novel enzyme discovery

The Archaea are a group of microorganisms that are phylogenetically distinct to the Bacteria and Eucarya. Their size and shape resemble bacteria, but display stark differences in the structure of their membrane lipids and machinery that are responsible for DNA replication and transcription. In addition, Archaea seem to utilize metabolic pathways that differ to previously recognized, classical pathways in bacteria and eukaryotes. Based on the genome sequences of the Archaea, there are many cases in which a particular metabolic pathway seems to be absent or incomplete. The search for these “missing” pathways or enzymes has been an exciting field of research in the Archaea, and has led to the discovery of structurally novel enzymes or enzymes with novel activity. Until now, we have been focused on the metabolism of the hyperthermophilic archaeon Thermococcus kodakarensis. The organism is an obligate anaerobe and heterotroph, utilizing a wide range of organic compounds including peptides/amino acids, starch and maltooligosaccharides, and organic acids such as pyruvate. By searching for missing genes, we have identified a structurally novel fructose-1,6-bisphosphatase [1], a key enzyme in gluconeogenesis, and enzymes with novel activity, such as pantoate kinase and phosphopantothenate synthetase, both involved in coenzyme A biosynthesis in T. kodakarensis [2]. A number of genes predicted to encode kinases are present on the T. kodakarensis genome. Although more than half display similarity to characterized enzymes high enough to predict their substrates, there are still nearly 20 genes whose substrates are unknown. One of them turned out to be a myo-inositol 3-kinase [3]. Another was found to display an ADP-dependent ribose-1-phosphate kinase activity, which participates in a pentose bisphosphate pathway, a previously unidentified route to direct the ribose moieties of nucleosides to central carbon metabolism [4]. Another kinase was identified through studies on serine and cysteine metabolism in T. kodakarensis. The protein was initially annotated as a chromosome-partitioning protein ParB, but displayed ADP-dependent serine kinase activity. The enzyme was necessary for the conversion of Ser to Cys in vivo, and is most likely involved in Ser assimilation in this archaeon [5]. The structure of the enzyme explains the specificity of the enzyme towards Ser and ADP, and raises the possibility that structurally related proteins may also be present not only in archaea but also in bacteria [6]. These studies indicate the potential of archaea as a source for novel enzyme discovery demonstrate various means to identify gene function through genome sequence information. [1] Rashid N, Imanaka H, Kanai T, Fukui T, Atomi H, Imanaka T. A novel candidate for the true fructose-1,6-bisphosphatase in archaea. J. Biol. Chem., 277(34):30649-30655, 2002. [2] Yokooji Y, Tomita H, Atomi H, Imanaka T. Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea. J. Biol. Chem., 284(41):28137-28145, 2009. [3] Sato T, Fujihashi M, Miyamoto Y, Kuwata K, Kusaka E, Fujita H, Miki K, Atomi H. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity. J. Biol. Chem., 288(29):20856-20867, 2013. [4] Aono R, Sato T, Imanaka T, Atomi H. A pentose bisphosphate pathway for nucleoside degradation in Archaea. Nat. Chem. Biol., 11(5):355-360, 2015. [5] Makino Y, Sato T, Kawamura H, Hachisuka SI, Takeno R, Imanaka T, Atomi H. An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism. Nat. Commun., 7:13446, 2016. [6] Nagata R, Fujihashi M, Kawamura H, Sato T, Fujita T, Atomi H, Miki K. Structural study on the reaction mechanism of a free serine kinase involved in cysteine biosynthesis. ACS Chem. Biol. Article ASAP, 2017. doi:10.1021/acschembio.7b00064

Isolation of the Gene Encoding Yeast Peroxisomal Isocitrate Lyase by a Combination of the Plaque Hybridization with Non-Radioactive Probes and the Amplification of Phages in a Small Scale

A genomic DNA encoding isocitrate lyase, one of peroxisomal enzymes, was successfully isolated from an n-alkane-utilizable yeast genomic library prepared in a λEMBL phage by a combination of the plaque hybridization with a non-radioactive, biotin labeled, cDNA and the amplification of the phages in a small scale. Three clones, partially overlapping, with sizes of about 11, 13 and 16 kbp respectively were finally obtained. The genomic Southern blot analysis using the biotin-labeled probe suggested the presence of one isocitrate lyase gene in the genomic DNA

Expression Cloning of Catalase Genomic Gene : Genomic DNA Expression Library of Candida boidinii in Saccharomyces cerevisiae

The genomic DNA expression library of a methylotrophic yeast, Candida boidinii (Kloeckera sp.) 2201, was prepared in Saccharomyces cerevisiae by the electroporation method. Five transformants harbouring a catalase gene were independently isolated with an anti-C. boidinii catalase antibody from this library. Furthermore, exhibition of catalase activity in these transformants demonstrated that C. boidinii genes could sufficiently function even in S. cerevisiae

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon

AbstractA RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization

A non-carboxylating pentose bisphosphate pathway in halophilic archaea

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1, 5-bisphosphate (R15P) isomerase and ribulose-1, 5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco. In this study, we identify a previously unrecognized nucleoside degradation pathway in halophilic archaea, composed of guanosine phosphorylase, ATP-dependent ribose-1-phosphate kinase, R15P isomerase, RuBP phosphatase, ribulose-1-phosphate aldolase, and glycolaldehyde reductase. The pathway converts the ribose moiety of guanosine to dihydroxyacetone phosphate and ethylene glycol. Although the metabolic route from guanosine to RuBP via R15P is similar to that of the pentose bisphosphate pathway in Thermococcales, the downstream route does not utilize Rubisco and is unique to halophilic archaea

A glycolipid glycosyltransferase with broad substrate specificity from the marine bacterium “Candidatus Pelagibacter sp.” Strain HTCC7211

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, nonphosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium “Candidatus Pelagibacter sp.” strain HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol (DAG) as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme’s affinity for UDP-glucose, which improves its catalytic efficiency. Homology modeling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling but also describe an efficient enzymatic tool for the future synthesis of bioactive molecules

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone-like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle-positioning code and are excluded from gene-regulatory DNA suggesting a functional organization. Beads-on-a-string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone-fold proteins in a variety of multimeric forms