Structures of the Bradyrhizobium japonicum FixL heme domain have been determined in the absence and presence of specific ligands to elucidate the detailed features of its O2 sensing mechanism. The putative roles of spin-state and steric hindrance were evaluated by the structure determination of ferrous CO-bound BjFixLH and correlating its features with other ligand-bound structures. As found for NO-BjFixLH, no protein conformational change was observed in CO-BjFixLH, suggesting a more complicated mechanism than solely spin state or ligand sterics. To evaluate the role of oxidation state, the structure of the ferrous deoxy-BjFixLH was determined. The structure of deoxy-BjFixLH was found to be virtually identical to the structure of the ferric met-BjFixLH. The role of hydrogen bonding of substrates to a heme-pocket water was evaluated by determining the structure of BjFixLH bound to 1-methyl-imidazole that cannot form a hydrogen bond with this water. In this case, the heme-mediated conformational change was observed, limiting the potential importance of this interaction. Finally, the structure of cyanomet-BjFixLH was revisited to rule out concerns regarding the partial occupancy of the cyanide ligand in a previous structure. In the revised structure, Arg 220 was found to move into the heme pocket to form a hydrogen bond to the bound cyanide ligand. The implications of these results on FixL's sensing mechanism are discussed.

The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.

The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.