Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 A resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (ST II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 A. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed.

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The crystal structure of native salmon pancreatic elastase (SPE) has been solved by molecular-replacement methods, and refined by conventional conjugate-gradient methods and simulated-annealing techniques. The final R value is 17.2% for 21 389 reflections between 8.0 and 1.61 A,, and the corresponding free R value is 23.9%. The overall tertiary structure of SPE is remarkably similar to that of porcine pancreatic elastase I (PPE), to which it shows about 67% sequence identity. The primary structure of SPE is determined from the electron-density maps, and only about 15 side chains are somewhat uncertain. Interesting differences between SPE and PPE, are one sequence deletion assigned to position 186, the residue 192 at the entrance of the specificity pocket is substituted from a Gin in PPE to Asn in SPE, and one of the calcium ligands is different. Furthermore, electron density is missing in SPE for the last three residues of the C-terminal helix. A comparison of the present aminoacid sequence of SPE with other sequences available indicates that SPE belongs to the class 1 pancreatic elastases.

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