Engineering Tk1656, a highly active l-asparaginase from Thermococcus kodakarensis, for enhanced activity and stability.

l-Asparaginases catalyze the hydrolysis of l-asparagine to l-aspartic acid and ammonia. These enzymes have potential applications in therapeutics and food industry. Tk1656, a highly active and thermostable l-asparaginase from Thermococcus kodakarensis, has been proved effective in selective killing of acute lymphocytic leukemia cells and in reducing acrylamide formation in baked and fried foods. However, it displayed <5 % activity under physiological conditions compared to the optimal activity at 85 °C and pH 9.5. We have attempted engineering of this valuable enzyme to improve the characteristics required for therapeutic and industrial applications. Based on the literature and crystal structure of Tk1656, nine specific mutant variants were designed, produced in Escherichia coli, and the purified mutant enzymes were compared with the wild-type. One of the mutants, K299L, displayed >20 % increase in activity at 85 °C. H158S substitution resulted in >5 °C increase in the optimal temperature. Similarly, a mesophilic-like mutation L56D, resulted in >5-fold increase in activity at pH 7.0 and 37 °C compared to that of the wild-type enzyme. The substrate specificity of the mutant variants remained unchanged. These results demonstrate that L56D and K299L variants of Tk1656 are the potent enzymes for therapeutics and acrylamide mitigation applications, respectively.

Structural and functional investigations of Pcal_0606, a bifunctional phosphoglucose/phosphomannose isomerase from Pyrobaculum calidifontis.

We are investigating the glycolytic pathway in Pyrobaculum calidifontis whose genome sequence contains homologues of all the enzymes involved in this pathway. We have characterized most of them. An open reading frame, Pcal_0606, annotated as a putative phosphoglucose/phosphomannose isomerase has to be characterized yet. In silico analysis indicated the presence of more than one substrate binding pockets at the dimeric interface of Pcal_0606. The gene encoding Pcal_0606 was cloned and expressed in Escherichia coli. Recombinant Pcal_0606, produced in soluble form, exhibited highest enzyme activity at 90 °C and pH 8.5. Presence or absence of metal ions or EDTA did not significantly affect the enzyme activity. Under optimal conditions, Pcal_0606 displayed apparent Km values of 0.33, 0.34, and 0.29 mM against glucose 6-phosphate, mannose 6-phosphate and fructose 6-phosphate, respectively. In the same order, Vmax values against these substrates were 290, 235, and 240 µmol min-1 mg-1, indicating that Pcal_0606 catalyzed the reversible isomerization of these substrates with nearly same catalytic efficiency. These results characterize Pcal_0606 a bifunctional phosphoglucose/phosphomannose isomerase, which displayed high thermostability with a half-life of ∼50 min at 100 °C. To the best of our knowledge, Pcal_0606 is the most active and thermostable bifunctional phosphoglucose/phosphomannose isomerase characterized to date.

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis.

The genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0039, which encodes a putative DNA ligase. Structural analysis disclosed the presence of signature sequences of ATP-dependent DNA ligases. We have heterologously expressed Pcal_0039 gene in Escherichia coli. The recombinant protein, majorly produced in soluble form, was purified and functionally characterized. Recombinant Pcal_0039 displayed nick-joining activity between 40 and 85 °C. Optimal activity was observed at 70 °C and pH 5.5. Nick-joining activity was retained even after heating for 1 h at 90 °C, indicating highly thermostable nature of Pcal_0039. The nick-joining activity, displayed by Pcal_0039, was metal ion dependent and Mg2+ was the most preferred. NaCl and KCl inhibited the nick-joining activity at or above 200 mmol/L. The activity catalyzed by recombinant Pcal_0039 was independent of addition of ATP or NAD+ or any other nucleotide cofactor. A mismatch adjacent to the nick, either at 3'- or 5'-end, abolished the nick-joining activity. These characteristics make Pcal_0039 a potential candidate for applications in DNA diagnostics. To the best of our knowledge, Pcal_0039 is the only DNA ligase, characterized from genus Pyrobaculum, which exhibits optimum nick-joining activity at pH below 6.0 and independent of any nucleotide cofactor.

Molecular cloning, expression in Escherichia coli and structural-functional analysis of a pyruvate kinase from Pyrobaculum calidifontis.

This manuscript describes recombinant production, characterization and structural analysis of wild-type and mutant Pcal_0029, a pyruvate kinase from Pyrobaculum calidifontis. Recombinant Pcal_0029 was produced in soluble and highly active form in Escherichia coli. Purified protein exhibited divalent metal-dependent activity which increased with the increase in temperature till 85 °C. Recombinant Pcal_0029 was highly thermostable with no significant loss in activity even after an incubation of 120 min at 100 °C. The enzyme exhibited apparent S0.5 and Vmax values of 0.44 ± 0.05 mM and 840 ± 39 units, respectively, towards phosphoenolpyruvate. These values towards adenosine-5'-diphosphate were 0.5 ± 0.07 mM and 870 ± 26 units, respectively. In silico structural analysis and comparison with the characterized enzymes revealed the presence of eight conserved regions. Two substitutions, K130E and S155G, resulted in a 10-fold decrease in activity. Secondary structure analysis indicated similar structures for the wild-type and the mutant enzymes. Bioinformatics analysis revealed disruption of interatomic interactions and hydrogen bonds, leading to a decreased flexibility and solvent accessibility, which may have led to decrease in activity. To the best of our knowledge, Pcal_0029 is the most thermostable pyruvate kinase reported so far. Moreover, this is the first study on the role of non-catalytic residues in a pyruvate kinase.

A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan.

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

Isocitrate dehydrogenase 1 gene variants analysis of glioma patients from Pakistan.

Various somatic isocitrate dehydrogenase 1 (IDH1) gene variants have been reported to drive lower-grade gliomas and secondary glioblastomas. In the current study, we explored the IDH1 variants in the glioma biopsy samples of patients from Pakistan. We explored the incidence of isocitrate dehydrogenase 1 gene variants by hotspot sequencing in 80 formalin-fixed paraffin-embedded tissues of different types of glioma biopsy samples. Structural modeling of the identified variants in isocitrate dehydrogenase 1 protein was done to see their possible consequences. The frequently described p.Arg132 variants were not found in any of the glioma types. However, in our study, we identified nonsynonymous variants at the residues p.R109 and p.G136 in astrocytomas and p.R100 in oligodendroglioma. These variants are affecting a part of the conserved domain in isocitrate dehydrogenase 1. Both of p.R100 and p.R109 variants are rare and described before, whereas the p.G136 variant identified in this study has never been described previously. Structural modeling showed that variants of these residues would directly affect the substrate binding and hence the enzyme activity.

Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis.

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 µmol min-1 mg-1 towards l-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards l- or d-glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 µmol min-1 mg-1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol-1. To the best of our knowledge, this is the first characterization of a plant-type l-asparaginase from class Thermococci of phylum Euryarchaeota.

Dengue proteins with their role in pathogenesis, and strategies for developing an effective anti-dengue treatment: A review.

Dengue virus is an arbovirus belonging to class Flaviviridae Its clinical manifestation ranges from asymptomatic to extreme conditions (dengue hemorrhagic fever/dengue shock syndrome). A lot of research has been done on this ailment, yet there is no effective treatment available for the disease. This review provides the systematic understanding of all dengue proteins, role of its structural proteins (C-protein, E-protein, prM) in virus entry, assembly, and secretion in host cell, and nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) in viral assembly, replication, and immune evasion during dengue progression and pathogenesis. Furthermore, the review has highlighted the controversies related to the only commercially available dengue vaccine, that is, Dengvaxia, and the risk associated with it. Lastly, it provides an insight regarding various approaches for developing an effective anti-dengue treatment.

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase).

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of ß-mercaptoethanol (ß-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of ß-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.

Merkel cell polyomavirus DNA sequences in the blood of healthy population of Pakistan.

Aim: This study aimed at detecting and quantifying Merkel cell polyomavirus (MCPyV) viral loads in the peripheral blood of healthy Pakistani individuals. Patients & methods: A total of 221 whole blood samples obtained from healthy individuals were examined by qPCR. Results & conclusion: MCPyV was detected in the peripheral blood of 31.2% healthy individuals. The rate of MCPyV positivity decreased from young (36%), to middle (33.7%) and elder (25.3%) age groups. Our data revealed higher prevalence of MCPyV in women (43.93%) than men (25.80%). The MCPyV viral load was calculated in the range of 0.06 -11 copies/ng of isolated DNA. The MCPyV viral load increased from young (median = 3.35) to elder (median = 5.66) age groups. The MCPyV circulate at a higher frequency by residing dormant in certain blood cells, which might act as potential vehicles for the spread of MCPyV infection among general population.

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses.

It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyomaviruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9.

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli.

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.

Structure and function of the thermostable L-asparaginase from Thermococcus kodakarensis.

L-Asparaginases catalyse the hydrolysis of asparagine to aspartic acid and ammonia. In addition, L-asparaginase is involved in the biosynthesis of amino acids such as lysine, methionine and threonine. These enzymes have been used as chemotherapeutic agents for the treatment of acute lymphoblastic leukaemia and other haematopoietic malignancies since the tumour cells cannot synthesize sufficient L-asparagine and are thus killed by deprivation of this amino acid. L-Asparaginases are also used in the food industry and have potential in the development of biosensors, for example for asparagine levels in leukaemia. The thermostable type I L-asparaginase from Thermococcus kodakarensis (TkA) is composed of 328 amino acids and forms homodimers in solution, with the highest catalytic activity being observed at pH 9.5 and 85°C. It has a Km value of 5.5 mM for L-asparagine, with no glutaminase activity being observed. The crystal structure of TkA has been determined at 2.18 Šresolution, confirming the presence of two α/ß domains connected by a short linker region. The N-terminal domain contains a highly flexible ß-hairpin which adopts `open' and `closed' conformations in different subunits of the solved TkA structure. In previously solved L-asparaginase structures this ß-hairpin was only visible when in the `closed' conformation, whilst it is characterized with good electron density in all of the subunits of the TkA structure. A phosphate anion resides at the active site, which is formed by residues from both of the neighbouring monomers in the dimer. The high thermostability of TkA is attributed to the high arginine and salt-bridge content when compared with related mesophilic enzymes.

Glutathione-Dependent Formaldehyde Dehydrogenase Homolog from Bacillus subtilis Strain R5 is a Propanol-Preferring Alcohol Dehydrogenase.

Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathione-dependent formaldehyde dehydrogenase.

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Genetic variants in FGFR2 and TNRC9 genes are associated with breast cancer risk in Pakistani women.

Single nucleotide polymorphisms (SNPs) lead to genetic differences in breast cancer (BC) susceptibility among women from different ethnicities. The present study aimed at investigating the involvement of SNPs of three genes, including fibroblast growth factor receptor 2 (FGFR2), trinucleotide-repeat-containing 9 (TNRC9) and mitogen-activated protein kinase kinase kinase 1 (MAP3K1), as risk factors for the development of BC. A case­control study (90­100 cases; 90­100 controls) was performed to evaluate five genetic variants of three genes, including FGFR2 (SNPs: rs1219648, rs2981582), TNRC9 (SNPs: rs8051542, rs3803662) and MAP3K1 (SNP: rs889312) as BC risk factors in Pakistani women. Significant associations were observed between BC risk and two SNPs of FGFR2 [rs2981582 (P=0.005), rs1219648 (P=9.08e­006)] and one SNP of TNRC9 [rs3803662) (P=0.012)] in Pakistani women. On examining the different interactions of these SNPs with various clinicopathological characteristics, all three associated genetic variants, rs2981582 rs1219648 and rs3803662, exhibited a greater predisposition to sporadic, in comparison to familial, BC. Furthermore, there was an increased effect of BC risk between haplotype combinations of the two SNPs of FGFR2 (rs2981582 and rs1219648) in Pakistani women. The results of the present study suggest that variants of FGFR2 and TNRC9 may contribute to the genetic susceptibility of BC in Pakistani women.

Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from Pyrobaculum calidifontis.

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.