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The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

In the crystal structure of the polyiodide complex (p-nitrophenyl-alpha-maltohexaose(2)) . Ba(I(3))(2) . 22H(2)O, the maltohexaose units form an antiparallel, left-handed double helix with O-2 ... O-3 and O-6 ... O-6 hydrogen bonding and a central cavity that encloses two triiodide units. This structure contrasts with the parallel, left-handed double helix with no central cavity proposed for the A-and B-starch helix and the left-handed single helix in V-amylose and may be relevant for the stabilization of glycogen Structure.

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).

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Three-phase partitioning is fast developing as a novel bioseparation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. Despite all this, the enzyme behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and the mixture was incubated at 25 degrees C for 1 h. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The specific activity of the processed enzyme was estimated and was found to be 210% of the original enzyme activity. In order to understand the basis of this remarkable enhancement of the enzyme activity, the structure of the TPP-treated enzyme was determined by X-ray diffraction at 1.5 A resolution. The overall structure of the TPP-treated enzyme is similar to the original structure in an aqueous environment. The hydrogen bonding system of the catalytic triad is intact. However, the water structure in the substrate binding site has undergone a rearrangement as some of the water molecules are either displaced or completely absent. Two acetate ions were identified in the structure. One is located in the active site and seems to mimic the role of water in the enzyme activity and stability. The other is located at the surface of the molecule and is involved in stabilizing the local structure of the enzyme. The most striking observation in respect of the present structure pertains to a relatively higher overall temperature factor (B = 19.7 A(2)) than the value of 9.3 A(2) in the original enzyme. As a result of a higher B-factor, a number of residues, particularly their side chains, were found to adopt more than one conformation. It appears that the protein exists in an excited state which might be helping the enzyme to function more rapidly than the original enzyme in aqueous media. Summarily, the basis of increased enzymatic activity could be attributed to (i) the presence of an acetate ion at the active site and (ii) its excited state as reflected by an overall higher B-factor.