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PubMed2005

Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment.

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.

DNA Transposable Elements↗

PubMed1994

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Amino Acid Sequence↗

PubMed1997

The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,.

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.

Amino Acid Sequence↗

PubMed1997

Biochemical characterisation of ornithine carbamoyltransferase from Pyrococcus furiosus.

Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400 +/- 20-kDa polymer of a 35-kDa subunit, in keeping with the corresponding gene sequence [Roovers, M., Hethke, C., Legrain, C., Thomm, M. & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur. J. Biochem. 247, 1038-1045]. In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P. furiosus OTCase exhibits no substrate cooperativity. In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function. Half-life estimates for the purified enzyme ranged over 21-65 min at 100 degrees C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate. The stability was not markedly influenced by the protein concentration. Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X-ray structure of P. aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers. The comparison between P. aeruginosa and P. furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry. Recombinant P. furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-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 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-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.

Amino Acid Sequence↗

PubMed1997

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).

Amino Acid Sequence↗

PubMed1998

Isoprenoid synthesis gene for geranylgeranyl diphosphate synthase from a hyperthermophile, Pyrococcus sp. strain OT3.

Pyrococcus sp. strain OT3, a hyperthermophilic archaeon that was isolated by the authors was found to contain tetraether lipid mainly in the membrane lipid, which was quite different from the other hyperthermophiles (Masuchi et al. 1997). Those isoprenoids are synthesized by a family of isoprenyl diphosphate synthases from isopentenyl diphosphate to allylic diphosphates. The gene that encodes one of these families, geranylgeranyl diphosphate synthase (GGPPSase), from this strain was cloned and sequenced. This coding gene has a 960-bp (320aa) sequence. The putative Shine-Dalgarno sequence was six bases upper of start codon, exactly the same as Methanobacterium thermoautotrophicum, a methnogenic thermophile. Comparison of the amino acid sequence of 13 organisms including Eukarya, Bacteria, and Archaea showed that Archaea strains including Pyrococcus sp. strain OT3 consisted of a separate group from the others, but five conservative regions are very homologous.

Journal Article↗

PubMed2004

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli.

A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector. One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate. The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P. furiosus. Expression of active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter. Assays of esterase activity in heat-treated extract of the recombinant E. coli showed the highest temperature optimum (100 degrees C) and thermostability (a half-life of 50 min at 126 degrees C) among esterases reported to date.

Cloning, Molecular↗

PubMed2001

Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus beta-glucosidase: effects of donor concentration, water content, and temperature on activity and selectivity in hexanol.

The transglucosylation reaction catalyzed by wild-type beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-beta-glucoside to hexyl-beta-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The selectivity towards transglucosylation was quantified by the S value defined as follows: S = r(S) x a(W)/r(H) x a(hex) where r(S) and r(H) are the initial rates of transglucosylation and hydrolysis and a(w) and a(hex) are the thermodynamic activities of water and hexanol. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-beta-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95 degrees C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-beta-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (a(w)), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as a(w) = 0.29. Surprisingly, the S value increased with increasing water activity up to a(w) = 0.92. At still higher water content the S value decreased.

Chromatography, High Pressure Liquid↗

PubMed2001

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.

Amino Acid Sequence↗

PubMed2005

Crystal structure and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3.

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.

Acid Anhydride Hydrolases↗

PubMed2005

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study.

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.

Bacterial Proteins↗

PubMed1999

Analysis of DNA compaction profile and intracellular contents of archaeal histones from Pyrococcus kodakaraensis KOD1.

Two histone genes, hpkA and hpkB, from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain were cloned, sequenced, and expressed in Escherichia coli cells. Both hpkA and hpkB genes encoded a protein of 67 amino acids, however they possessed the different molecular weight (HpkA, 7,378:HpkB, 7,167). Deduced amino acid sequences of HpkA and HpkB were homologous to other archaeal histones and eucaryal core histones (H2A, H4). Gel mobility shift assays by purified proteins demonstrated that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA. HpkB prevented double stranded DNA from thermal denaturation in less amount than HpkA in vitro. In order to investigate intracellular contents of HpkA and HpkB in KOD1 cells, immunoblot analysis was performed by using anti-HpkA antisera obtained from immunized BALB/c mice, showing that HpkA was less abundantly expressed than HpkB in KOD1 cells. These results suggest that HpkB plays a major role to protect double stranded DNA from thermal denaturation in vivo.

Amino Acid Sequence↗

PubMed2002

Active site of deblocking aminopeptidase from Pyrococcus horikoshii.

New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion.

Amino Acid Sequence↗

PubMed1998

Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus.

The structure of methionine aminopeptidase from hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100 degreesC was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A. The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement. This is the first description of a eukaryotic type methionine aminopeptidase structure. The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel beta-strands. The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase. Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel beta-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues.

Amino Acid Sequence↗

PubMed1999

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus: the 1.85 A resolution crystal structure and its mechanistic implications.

Crystal structures of formaldehyde ferredoxin oxidoreductase (FOR), a tungstopterin-containing protein from the hyperthermophilic archaeon Pyrococcus furiosus, have been determined in the native state and as a complex with the inhibitor glutarate at 1.85 A and 2. 4 A resolution, respectively. The native structure was solved by molecular replacement using the structure of the homologous P. furiosus aldehyde ferredoxin oxidoreductase (AOR) as the initial model. Residues are identified in FOR that may be involved in either the catalytic mechanism or in determining substrate specificity. The binding site on FOR for the physiological electron acceptor, P. furiosus ferredoxin (Fd), has been established from an FOR-Fd cocrystal structure. Based on the arrangement of redox centers in this structure, an electron transfer pathway is proposed that begins at the tungsten center, leads to the (4Fe:4S) cluster of FOR via one of the two pterins that coordinate the tungsten, and ends at the (4Fe:4S) cluster of ferredoxin. This pathway includes two residues that coordinate the (4Fe:4S) clusters, Cys287 of FOR and Asp14 of ferredoxin. Similarities in the active site structures between FOR and the unrelated molybdoenzyme aldehyde oxidoreductase from Desulfovibrio gigas suggest that both enzymes utilize a common mechanism for aldehyde oxidation.

Aldehyde Oxidoreductases↗

PubMed1999

Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus.

The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli. The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps. The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours). pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe). It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups. Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product. The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions. Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast. The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule. These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group. In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule.

Cloning, Molecular↗