We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at ؊5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca 2؉ . Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca 2؉ -dependent lipase. Addition of Mn 2؉ and Sr 2؉ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca 2؉ . The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C 10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.Lipases (glycerol ester hydrolases) are hydrolases acting on the carboxyl ester bonds present in acylglycerols to liberate fatty acids and glycerols. Lipases are versatile enzymes that are distributed throughout living organisms. A vast number of bacterial lipases with different enzymological properties and substrate specificities have been found (20). They have a wide range of potential applications in the hydrolysis, esterification, and transesterification of triglycerides and in the chiral selective synthesis of esters (17,19
Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp Tk ) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, Imp Tk , was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed ⌬fbp and ⌬imp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The ⌬fbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbp Tk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous imp Tk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that imp Tk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the Fbp Tk ortholog, not the IMPase/FBPase ortholog.
A hyperthermophilic archaeon strain, KODI, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100°C, and the optimal temperature was 950C. The anaerobic strain was an So-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110°C, with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90°C for 2 h, and the half-life of enzymatic activity at 100°C was 60 min. The proteolytic activity was significantly inhibited byp-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.
Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway. Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities. Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M. jannaschii, displays high FBPase activity. The FBPase from strain KOD1 was partially purified, its Nterminal amino acid sequence was determined, and the gene (Tk-fbp) was cloned. Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da. The recombinant Tk-Fbp was purified and characterized. Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K m value of 100 M toward fructose 1,6-bisphosphate, and a k cat value of 17 s ؊1 subunit ؊1 at 95°C. Unlike the inositol monophosphatase from M. jannaschii, Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate. Activity was enhanced by Mg 2؉ and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate. AMP did not inhibit the enzyme activity. We examined whether expression of Tk-fbp was regulated at the transcription level. High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium. Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M. jannaschii, Pyrococcus furiosus, Sulfolobus solfataricus, and Archaeoglobus fulgidus, but are yet to be assigned any function. Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea.Glycolysis and gluconeogenesis are pathways involved in the degradation and synthesis of intracellular sugars, respectively.
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