Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

POLLUX: a program for simulated cloning, mutagenesis and database searching of DNA constructs.

Computer support for research in biotechnology has developed rapidly and has provided several tools to aid the researcher. This report describes the capabilities of new computer software developed in this laboratory to aid in the documentation and planning of experiments in molecular biology. The program, POLLUX, provides a graphical medium for the entry, edit and manipulation of DNA constructs and a textual format for display and edit of construct descriptive data. Program operation and procedures are designed to mimic the actual laboratory experiments with respect to capability and the order in which they are performed. Flexible control over the content of the computer-generated displays and program facilities is provided by a mouse-driven menu interface. Programmed facilities for mutagenesis, simulated cloning and searching of the database from networked workstations are described.

Three-dimensional structure of a genetically engineered variant of porcine growth hormone.

The three-dimensional structure of a genetically engineered variant of porcine growth hormone, methionyl porcine somatotropin (MPS), has been determined at 2.8-A resolution, using single crystal x-ray diffraction techniques. Phases were obtained by use of a single isomorphous K2OsCl6 derivative and were improved by use of the density modification procedure. The MPS structure is predominantly helical. It consists mainly of four antiparallel alpha-helices arranged in a left twisted helical bundle, a structural motif observed in a number of other unrelated proteins. However, the way the four helices are connected in the bundle is unusual and, to our knowledge, has never been reported before. Alignment of the amino acid sequence of MPS with that of other growth hormones reveals that residues within the alpha-helices are predominantly invariant and thus these invariant residues are necessary to maintain the structural integrity of these proteins.