Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 A, 1.48 A and 2.01 A, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 A over all Calpha positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.
Conversion of pimeloyl-coenzyme A (CoA) to biotin in Escherichia coli requires at least four enzymes encoded by genes in the bio operon. One gene, bioH, which is not present in the bioABFCD operon, is required for the synthesis of pimeloyl-CoA but its exact role in formation of this intermediate is unknown. To investigate this further, we have overexpressed and purified the bioH gene products from both E. coli (BIOH EC) and Neisseria meningitis (BIOH NM) in E. coli. When purified BIOH was incubated with excess CoA and analysed by electrospray mass spectrometry a species of mass corresponding to a BIOH:CoA complex was observed. Mutation of a conserved serine residue to alanine (BIOH EC S82A) did not prevent CoA binding. This is the first report of the purification of BIOH and the observation of a small molecule bound to the protein provides clues to its role in pimeloyl-CoA synthesis.