The open conformation of a Pseudomonas lipase.

BACKGROUND: . The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases. RESULTS: . The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site. CONCLUSIONS: . The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

Crystallographic investigations of subtilisin BPN' mutants engineered for studying thermal stability.

Subtilisin BPN' (BPN) is an industrially important serine protease that has been extensively investigated in many laboratories. In an effort to improve the thermal stability of the enzyme, researchers at Procter & Gamble have used site-directed mutagenesis techniques to produce several variants of BPN in which residues at the surface of the enzyme have been substituted. We initiated crystallographic studies to determine the structural consequences of these amino acid substitutions. In the course of this work we obtained excellent crystals that correspond to the C2 crystal form of native BPN that has been previously reported. Since the structure reported in that work was of only medium resolution, high-resolution X-ray data for this crystal form of native BPN have been collected and the refinement of the structure has been extended using these new data. Isomorphous crystals of two variants, Q19E and Q271E, have also been grown, high-resolution X-ray data have been collected for these crystals, and the experimental results are described. The structures of the native enzyme and the Q271E variant have been refined and are described.

Purification, crystallization and preliminary X-ray investigation of aqualysin I, a heat-stable serine protease.

Aqualysin I, a thermostable protease found in the culture medium of Thermus aquaticus YT-1, has been purified to homogeneity using a combination of ion-exchange and affinity chromatography. It is a polypeptide with a molecular weight of 28 350 [Kwon, Terada, Matsuzawa & Ohta (1988). Eur. J. Biochem. 173, 491-497] and is most active at 343-353 K and pH about 10.0 [Matsuzawa, Tokugawa, Hamaoki, Mizoguchi, Taguchi, Terada, Kwon & Ohta (1988). Eur. J. Biochem. 171, 441-447]. Crystals of the enzyme are monoclinic, space group P2(1), with cell dimensions a = 40.80 (5), b = 64.39 (6), c = 45.51 (6) A and beta = 109.1 (1) degrees. The asymmetric unit consists of a single molecule (V(m) = 1.99 A(3)Da(-1)). The crystals are stable to X-radiation and scatter to at least 2.8 A resolution.