Well diffracting crystals of rhamnogalacturonan acetylesterase from Aspergillus aculeatus have been obtained in two polymorphic modifications despite its heterogeneous glycosylation. The best-diffracting crystals (resolution 1.55 A) are orthorhombic. The limit of the diffraction pattern of the other (trigonal) form is 2.5 A. The ability of the enzyme to crystallize appears to depend on the glycosylation of the protein sample. This aspect has been investigated by mass spectrometry, which also showed that the orthorhombic crystals have the same glycosylation as the protein sample used in the crystallization.

Ascaris suum is an intestinal endoparasitic nematode found in pigs. In order to avoid proteolytic digestion by the host the nematode has developed a large battery of proteinase inhibitors. Among those is the aspartic proteinase inhibitor PI-3. PI-3 consists of a single polypeptide chain containing 149 residues with a molecular weight of 16.4 kD. It has three disulfide bridges. CD and secondary structure predictions indicate that the fold of PI-3 is mixed alpha/beta.1 Until now only a very few larger aspartic proteinase inhibitors have been characterized. Aspartic proteinase inhibitors from the Filarie family of parasites have been reported (Dirofilaria, Brugia and Onchocerca). These inhibitors are more closely related to each other (~60% sequence identity) than PI-3 (less than 20%).2 The inhibitor form Ascaris suum show a broad specificity towards members from the pepsin related family of aspartic proteinases having Ki’s in the range of 0.2—15 nM (porcine and human pepsin and cathepsin E). Cathepsin D however, is not inhibited by PI-3, although it shares up to 45 identity to the above mentioned aspartic proteinases.3 This has important aspects as it might be used to distinguish between cathepsin D and E. The following report describes the progress in the structure determination of the molecular complex between porcine pepsin and PI-3.

The structure of a recombinant peroxidase from the ink cap, Coprinus cinereus, CiP, is reported to 2.6 Å resolution and refined to a R‐value of 18.1%. The structure was solved by molecular replacement using the coordinates from a newly published ligninase structure, LiP. CiP crystallizes in space group P212121) with two independent molecules in the asymmetric unit related by the vector 0.29b + 0.5C. The two CiP molecules are structurally identical; each contains two Ca2+ ions in positions equivalent to those found in the LiP structure. Two N‐acetylglucosamines and one mannose residue were fitted into the density adjacent to two of the three predicted glycosylation sites. The refinement also included 40 and 41 water molecules, respectively, in the two CiP molecules. The structure of CiP displays a folding very similar to that of LiP. The active sites are almost identical in the LiP and CiP structures. CiP has a much larger opening to the active site than LiP.

BACKGROUND:The trefoil peptides are a rapidly growing family of peptides, mainly found in the gastrointestinal tract. There is circumstantial evidence that they stabilize the mucus layer, and may affect the rate of healing of the mucosal epithelium. RESULTS:We have determined the structure of porcine pancreatic spasmolytic polypeptide (PSP) to 2.5 A resolution. The polypeptide contains two trefoil domains. The domain structure is compact, and is composed of a central short antiparallel beta-sheet with one short helix above and one below it. This is a novel motif. The two domains are related by two-fold symmetry, and each domain contains a cleft. CONCLUSIONS:The cleft within each domain could accommodate a polysaccharide chain, and may therefore be responsible for binding mucin glycoproteins. We suggest that PSP may cross-link glycoproteins, explaining its ability to stabilize the mucus layer.

Crystals suitable for an X-ray diffraction investigation have been obtained of recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae. The crystals were grown by the hanging drop method with polyethylene glycol 6000 as the precipitant. A pH range from 6·2 to 8·0 and CaCl2 or MgCl2 present at a concentration of 0·35 M were essential for the crystal growth. A metastable monoclinic modification can be obtained under certain conditions, and with variations in temperature they are transformed into a stable orthorhombic modification. With CaCl2 as the additive, the unit cell dimensions were a = 74·9 Å, b = 76·8 Å and c = 128·2 Å. With two peroxidase molecules per asymmetric unit, the solvent content is 49% (v/v). In the diffraction pattern, the reflections Okl are systematically very weak for k = 2n + 1. Combined with an analysis of the Patterson function, this showed that the two independent molecules are related by the pseudo-translational symmetry 0·29a+0·5b. The possible space groups are P 212121 or P 21221 because of this pseudosymmetry. The crystals diffract to a resolution of 2·9 Å.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P212121 with unit cell dimensions a = 62.95 Å, b = 66.24 Å and c = 70.78 Å. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 Å.