1. Tryptophan synthase We have continued work on the high resolution structure of the bifunctional enzyme complex tryptophan synthase. This complex is a paradigm for the study of intersubunit communication and allosteric activity. In the presence of indole,we have demonstrated the opening of a gate in the tunnel that is believed to channel indole between the two active sites. A series of conformational differences have been observed that provide a basis for understanding the mechanism of action and the communication between the two active sites. In the presence of substrate at both active site a series of changes occur that prevent solvent access to the active sites and ensure that efficient catalysis occurs.2. Deoxyhyposine synthase The crystal structure of the enzyme Deoxyhypusine synthase has been determined at 2.2A resolution. The enzyme is a homotetramer consisting of two pairs of closely linked subunits with the active sites shared between the subunits. The active site is located in a deep cavity in which the spermidine is presumably bound. The nicotinamide ring is oriented in a manner consistent with the established stereospecificity of the hydride reaction. One interesting feature of the structure is that there is an N-terminal helix from a third subunit that forms a ball and chain arrangement which is located so as to block access to the active site. A new crystal form of the enzyme reveals that the access to the active site pocket is now open with the N-terminal helices disordered. Spermidine analog inhibitors have been demonstrated to bind in the pocket.3. Serotonin N-acetyltransferase This enzyme is penultimate in the conversion of serotonin to melatonin, the nocturnal neurohormone produced in the pineal gland. The structure of ovine NAT has been determined at 2.5A resolution. The protein consists of a central beta sheet flanked by alpha-helices. Two parallel strands in the centerof the beta-sheet are pulled apart at their C-terminal ends, and form a "V"-shaped binding site for the cofactor, acetyl coenzyme A. This permits the identification of two histidine residues that could act as catalytic bases; furthermore, site directed mutagenesis results indicate that conservative mutation of either of these residues renders the protein inactive. On the basis of this structure a detailed catalytic mechanism has been proposed which is now in the process of being tested through mutagenic and kinetic analyses. We have also solved the high resolution structure of this enzyme complexed with a bisubstrate inhibitor. Surprisingly, upon binding the enzyme undergoes a major conformational change. This change provides an obvious structural explanation for previously documented kinetic properties of this enzyme. 4. Tn7 Transposon The Tn7 transposon is a mobile DNA element that moves from DNA site to DNA site using a complex array of self-encoded proteins. The transposon carries genes encoding for enzymes that confer resistance to trimethoprim and the aminoglycosides streptomycin and spectinomycin, and exemplifies one method by which multi-drug resistance can be transferred between organisms. One of the proteins which forms part of the transposase, TnsA, has recently been crystallized in the presence of its cofactor, Mg++, and native data have been collected to 2.4A. Contrary to expectations, the structure is not homologous to other transposases; instead it is similar to type II restriction endonucleases.