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A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

The target of diphtheria toxin is the diphthamide residue in translation elongation factor 2 (EF-2), which is generated by a three-step post-translational modification of a specific histidine residue in the EF-2 precursor. In the second modification step, an S-adenosylmethionine-dependent methyltransferase, diphthine synthase (DS), catalyzes the trimethylation of the EF-2 precursor. The homodimeric crystal structures of the archaeal diphthine synthases from Pyrococcus horikoshii OT3 and Aeropyrum pernix K1 have been determined. These structures share essentially the same overall fold as the cobalt-precorrin-4 methyltransferase CbiF, confirming that DS belongs to the dimeric class III family of methyltransferases. In the P. horikoshii DS dimer, only one of the two active sites binds the reaction product S-adenosyl-L-homocysteine (AdoHcy), while the other active site contains no ligand. This asymmetric AdoHcy binding may be a consequence of intra-domain and inter-domain movements upon binding of AdoHcy at one of the two sites. These movements disrupt the twofold dimeric symmetry of the DS dimer and probably cause lower AdoHcy affinity at the other binding site.

Aspartate racemase from Pyrococcus horikoshii OT3 (P. AspR) has been crystallized in three crystal forms by the sitting-drop vapour-diffusion method. The crystals belong to the space groups P2(1), P2(1)2(1)2(1) and P3(1)21 (or P3(2)21). The crystals of space group P2(1) diffract X-rays beyond 1.7 A resolution under 90 K liquid-nitrogen cryoconditions with synchrotron radiation and were selected for structure determination. Two heavy-atom derivatives of this crystal form were obtained by the soaking method, which afforded the initial electron-density map.

The archaeosine tRNA-guanine transglycosylase from the hyperthermophilic archaeon Pyrococcus horikoshii was crystallized and preliminary X-ray characterization was performed. Single crystals were grown by the hanging-drop vapour-diffusion method, using sodium/potassium phosphate and sodium acetate as precipitants. The space group is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 99.28 (14), c = 363.74 (56) A. The cryocooled crystals diffracted X-rays beyond 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8 (Harima). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.

Intein homing endonucleases are proteins spliced out from a precursor protein and site-specific enzymes that make double-strand breaks in inteinless alleles. Crystals of intein homing endonuclease II from the hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 (PI-PkoII) have been grown at room temperature using ammonium sulfate as a precipitant. The diffraction pattern of the crystal extends to 3.0 A resolution at room temperature upon exposure to synchrotron X-rays at KEK-PF, Japan. The crystals have symmetry consistent with space group C222(1), with unit-cell parameters a = 107.6, b = 150.5, c = 146.8 A. A full set of X-ray diffraction data were collected to 3.0 A Bragg spacing from a native crystal with an overall R(merge) of 4.8% and a completeness of 96.6%.

Crystals of archaeal O6-methylguanine-DNA methyltransferase (MGMT) from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 have been grown at room temperature using polyethylene glycol as a precipitant. The diffraction pattern of the crystal extends to 2.0 A resolution at room temperature upon exposure to Cu Kalpha radiation. The crystal belongs to the space group P212121 with unit-cell dimensions of a = 52.8, b = 86.6 and c = 39.9 A. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (Vm) of 2.3 A3 Da-1 and a solvent content of 48% by volume. A full set of X-ray diffraction data was collected to 2.0 A Bragg spacings from the native crystal.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.