Naeem Rashid

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

ABSTRACT 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 fromPseudomonas 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 Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. 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 C10 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.

Genetic Evidence Identifying the True Gluconeogenic Fructose-1,6-Bisphosphatase in Thermococcus kodakaraensis and Other Hyperthermophiles

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 (FbpTk) 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, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Deltafbp and Deltaimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Deltafbp 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, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous impTk 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 impTk 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 FbpTk ortholog, not the IMPase/FBPase ortholog.

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.

Presence of a Novel Phosphopentomutase and a 2-Deoxyribose 5-Phosphate Aldolase Reveals a Metabolic Link between Pentoses and Central Carbon Metabolism in the Hyperthermophilic Archaeon Thermococcus kodakaraensis

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.

A RecA / RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

Abstractu2002A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA). The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini. The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity. Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E. coli recA mutant strain.

Among Multiple Phosphomannomutase Gene Orthologues, Only One Gene Encodes a Protein with Phosphoglucomutase and Phosphomannomutase Activities in Thermococcus kodakaraensis

Four orthologous genes (TK1108, TK1404, TK1777, and TK2185) that can be annotated as phosphomannomutase (PMM) genes (COG1109) have been identified in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. We previously found that TK1777 actually encodes a phosphopentomutase. In order to determine which of the remaining three orthologues encodes a phosphoglucomutase (PGM), we examined the PGM activity in T. kodakaraensis cells and identified the gene responsible for this activity. Heterologous gene expression and purification and characterization of the recombinant protein indicated that TK1108 encoded a protein with high levels of PGM activity (690 U mg(-1)), along with high levels of PMM activity (401 U mg(-1)). Similar analyses of the remaining two orthologues revealed that their protein products exhibited neither PGM nor PMM activity. PGM activity and transcription of TK1108 in T. kodakaraensis were found to be higher in cells grown on starch than in cells grown on pyruvate. Our results clearly indicate that, among the four PMM gene orthologues in T. kodakaraensis, only one gene, TK1108, actually encodes a protein with PGM and PMM activities.

Highly Thermostable l-Threonine Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis

l-Threonine dehydrogenase, a key enzyme in the l-threonine metabolism, catalyses the NAD(+)-dependent conversion of l-threonine to 2-amino-3-ketobutyrate, that non-enzymically decarboxylates to aminoacetone. A search of the genome sequence of hyperthermophilic archaeon, Thermococcus kodakaraensis revealed the presence of a closely related orthologue (TK0916) of archaeal and bacterial l-threonine dehydrogenase genes. Expression in Escherichia coli, purification and characterization of the TK0916 gene product revealed that this gene actually coded for a protein with high levels of l-threonine dehydrogenase activity (7.26 U mg(-1)). The enzyme exhibited highest activity at pH 12 and 90 degrees C. The K(m) values for l-threonine and NAD(+) at 50 degrees C were 1.6 mM and 0.028 mM, respectively. The enzyme activity was dependent on divalent cations. The half-life of the enzyme was more than 2 h at 85 degrees C and 24 min in boiling water. This is the most thermostable threonine dehydrogenase exhibiting optimal activity at the highest pH (12) reported to date. This is the first report on the characterization of a TDH from genus Thermococcus.

An abnormally acidic TATA-binding protein from a hyperthermophilic archaeon

The gene encoding the TATA-binding protein (PkTBP) from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned and sequenced. An open reading frame with homology to the conserved C-terminal core region of eukaryotic TBP was expressed in Escherichia coli. Specific DNA-binding activity of the recombinant PkTBP (190 amino acids, 21.36 kDa) was also demonstrated. Although it was composed of a structurally direct repeat sequence which is similar to eukaryotic TBP, the total net charge of archaeal TBP was amazingly negative (calculated isoelectric point (pI) was 4.66 and experimentally estimated pI was 4.8). A series of five Glu residues was found at the C terminus of archaeal TBP. These data strongly suggest that a positively charged protein is also involved in the transcription initiation event which might stabilize the structure of the genomic DNA under high-growth-temperature conditions.

Isolation and characterization of psychrotrophs from subterranean environments

Subterranean environments are potential sources for the isolation of novel microorganisms. Water and soil samples were collected at depths ranging from 10 to 1800 meters below the surface, and screening was carried out with aerobic rich and anaerobic minimal media. Two psychrotrophic and three chemoautotrophic strains were isolated. One of the psychrotrophic isolates, designated SN16A, grew at temperatures between -5 and 37 degrees C with optimal growth between 25 and 30 degrees C. The other psychrotroph, designated KB700A, grew between -10 and 30 degrees C. Little difference in growth rate could be observed between 20 and 30 degrees C; however, this strain did not grow at 37 degrees C. KB700A utilized CO2 chemoautotrophically at 30 degrees C, using hydrogen as an energy source. Both strains were characterized biochemically. The complete 16S rRNA sequence of KB700A was 98.7% homologous with that of Pseudomonas marginalis. However, the 16S rRNA of SN16A showed only 95.4% identity at maximum-with the corresponding gene of Arthrobacter globiformis-suggesting that this strain may belong to a novel genus. Both strains exhibited the ability to produce hydrolytic enzymes on plate assays. Our results suggest that subterranean environments are promising sources for the isolation of psychrotrophic microorganisms.

Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.