Study of two proteins involved in protein disulphide formation : molecular cloning and characterization of a full-length flavin-dependent monooxygenase from Saccharomyces cerevisiae & preliminary structure analysis on DsbC from Haemophilus influenzae
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This work includes biochemistry studies on yeast FMO and structure analysis on a prokaryotic protein disulphide isomerase - H. influenzae DsbC. The yeast FMO (yFMO) gene was cloned, expressed and characterized in this lab previously. Deletion experiments suggested that yFMO was involved in folding proteins with disulfide bonds. In continuation of study on yeast FMO, we detected two nucleotide errors in the GenBank sequences of the yFMO gene. These errors had been incorporated into the initial gene engineering, and as a consequence, the protein studied initially is a truncated version of yFMO. To be consistent with previous work, the N-terminal his-tagged full-length yFMO was compared to the truncated enzyme in vitro. Using an oxygen uptake assay, the full-length and the truncated FMOs showed similar pH profiles, Km, Kcat and Vmax values using glutathione as a substrate, indicating that they share similar enzymatic character. Therefore, we conclude that the hypotheses proposed earlier concerning the enzyme’s functions are likely to be still valid for the full-length yeast FMO. The second project aimed for determining the structures of DsbC from several species using X-ray crystallography to improve our knowledge on the structure-function relationship of DsbC, and subsequently provide guidance for engineering DsbC to improve E. coli as a better protein expression system for proteins with multiple disulphide bonds. DsbC proteins from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Vibrio cholerae and Yersinia pseudotuberculosis were subjected to crystallization efforts, but only crystals of DsbC from H. inf and Y.pse have been obtained. A data set at the resolution of 2.5 Å was collected from a single H. inf DsbC crystal. Molecular replacement strategy was applied, using the known structure of E. coli DsbC, to solve the structure of H. inf DsbC. Due to a flexible protein conformation, many residue side chains in the N-terminal domain of the current structural model are missing. Comparison between the current H. inf DsbC model and E. coli DsbC structure shows that the H. inf DsbC has a similar catalytic domain and a larger, more open cleft within the V-shaped dimer.