Crystal structure of amylomaltase from thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans

Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the alpha-amylase superfamily of enzymes, whose core structure consists of a (beta, alpha)(8) barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the alpha-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the alpha-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the alpha-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the alpha-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.     

Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of alpha-1,4 glucans to produce cyclic alpha-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.     

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.     

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase

We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.     

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.     

The crystal structure of the Thermus aquaticus DnaB helicase monomer

The ring-shaped hexameric DnaB helicase unwinds duplex DNA at the replication fork of eubacteria. We have solved the crystal structure of the full-length Thermus aquaticus DnaB monomer, or possibly dimer, at 2.9Å resolution. DnaB is a highly flexible two domain protein. The C-terminal domain exhibits a RecA-like core fold and contains all the conserved sequence motifs that are characteristic of the DnaB helicase family. The N-terminal domain contains an additional helical hairpin that makes it larger than previously appreciated. Several DnaB mutations that modulate its interaction with primase are found in this hairpin. The similarity in the fold of the DnaB N-terminal domain with that of the C-terminal helicase-binding domain (HBD) of the DnaG primase also includes this hairpin. Comparison of hexameric homology models of DnaB with the structure of the papillomavirus E1 helicase suggests the two helicases may function through different mechanisms despite their sharing a common ancestor.     

The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

Crystallization and preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1

Many attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from thermus aquaticus. The crystals belong to hexagonal space group P6(3)(22) and diffract up to 2.6 A. The cell dimensions are a = b = 178 A, c = 238 A and 6 molecules are contained per asymmetric unit.     

Electron image analysis of ribosomal subunits from Thermus aquaticus.

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.     

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.     

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.2 X 10(-7) per recipient. Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings. Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.     

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus.

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.     

Determination of the structure of a novel glycolipid from Thermus aquaticus 15004 and demonstration that hydroxy fatty acids are amide linked to glycolipids in Thermus spp.

The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.     

Purification and characterization of a repressible alkaline phosphatase from Thermus aquaticus.

A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus. The enzyme can be derepressed more than 1,000-fold by starving the cells for phosphate. In derepressed cells, nearly 6% of the total protein in a cell-free enzyme preparation is alkaline phosphatase. The enzyme was purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. By sucrose gradient centrifugation it was established that the enzyme has an approximate molecular weight of 143,000 and consists of three subunits, each with a molecular weight of 51,000. Tris buffer stimulates the activity of the enzyme, which has a pH optimum of 9.2. The enzyme has a broad temperature range with an optimum of 75-80 degrees. The enzyme catalyzes the hydrolysis of a wide variety of phosphorylated compounds as do many of the mesophilic alkaline phosphatases. The Michaelis constant(Km) for the enzyme is 8.0 X 10(-4) M. Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.     

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.     

Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus.

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.     

X-ray structure of a dihydropyrimidinase from Thermus sp. at 1.3 A resolution

Dihydropyrimidinases (hydantoinases) catalyse the reversible hydrolytic ring-opening of cyclic diamides such as dihydropyrimidines in the catabolism of pyrimidines. In biotechnology, these enzymes find application in the enantiospecific production of amino acids from racemic hydantoins. The crystal structure of a D-enantio-specific dihydropyrimidinase from Thermus sp. (D-hydantoinase) was solved de novo by multiwavelength anomalous diffraction phasing. In spite of a large unit cell the D-hydantoinase crystals exhibit excellent diffraction properties. The structure was subsequently refined at 1.30 A resolution against native data. The core of D-hydantoinase consists of a (alpha/beta)(8)-barrel, which is flanked by a beta-sheet domain and some additional helices. In the active site, a carboxylated lysine residue and the catalytically active hydroxide ion bridge a binuclear zinc centre. The tertiary structure and shape of the active site show strong homology to that of ureases, dihydroorotases, and phosphotriesterases. The homology of the active site was exploited for in silicio docking of substrates in the active site. This could shed light both on the substrate binding in hydantoinases and on the recently highly discussed origin of the proton in the course of hydantoinase catalysis.