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The non-enzymatic deamidation of asparagine residues in proteins is a widely occurring reaction, both in vivo and in vitro. Although the importance of this process is commonly recognised, only little structural information is available on it. In order to evaluate the structural effects of this reaction in proteins, we have determined the crystal structure of a ribonuclease A derivative in which asparagine 67 has been replaced by an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. The overall structure of the model, refined to a crystallographic R-factor of 0.159 at a resolution of 1.9 A, is very similar to that of the native protein, but considerable deviations are observed in the region delimited by the disulphide bridge 65-72. In particular, the insertion of an extra methylene group in the main chain at residue 67 breaks up the hydrogen bond network that makes this region rather rigid in ribonuclease A. On the basis of the structure observed, some of the slightly but significantly different properties of this deamidated derivative, with respect to the native enzyme, can be explained.

The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.

An NAD + -dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized in the holo-enzyme and apo-enzyme forms. Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8·4 with the addition of NADH and at pH 7·0 with the addition of NADH and dimethyl sulphoxide. Crystals of the apo-enzyme grow at pH 6·3 from a mixture of polyethylene glycol 4000 and propan-2-ol. The holo-enzyme crystallizes in C 2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection. The apo-enzyme crystallizes in I 4 1 22 ( a = 126·82 A, b = 118·95 A) with a monomer in the asymmetric unit, and the single crystals diffract to 2·8 A.