| The E. coli medium chain-length acyl-CoA thioesterase II (TEII) is a representative of a novel and ubiquitous family of thioesterases. To date, its homologues have been found in bacteria, yeast and human. The human homologue has been identified in T-cells, and moreover was shown to bind to, and be activated by, the HIV Nef protein, suggesting a possible role in AIDS pathogenesis.;This dissertation describes the X-ray crystal structure of TEII and probing of its catalytic mechanism. The enzyme was subcloned, expressed, purified and crystallized. Due to the severe non-isomorphism among the native crystals, the multiwavelength anomalous dispersive (MAD) method was used to determine the structure of TEII from a single seleno-methionine labeled crystal at 2.5A resolution. The structure was then refined to 1.9A resolution with Rwork 21.8% and Rfree 24.8%. TEII has a novel fold consisting of a twelve-stranded antiparallel beta-sheet wrapped around two long central helices. The structure rules out the involvement of His58 in catalysis as suggested by chemical labeling, even though it is part of a trypsin-like triad. The new active site, which was proposed from the structure and supported by mutagenesis data, shows a novel chemistry for a thioesterase and contains a hydrogen bonding network of residues Ser 203, Asp204, Thr228, His231 and Gln278. Asp204 polarizes a water molecule for a nucleophilic attack on the substrate. TEII is the first structure obtained for this family of enzymes and it now sets the stage for further analysis of the structure-function relationship of TEII and its homologues. |
