Kinetic studies on the function of all the conserved tryptophans involved inside and outside the QW motifs of squalene-hopene cyclase: stabilizing effect of the protein structure against thermal denaturation.
Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC). Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs. However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6. Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases. On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation. The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure. It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids. The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0.
Sato
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Graduate School of Science and Technology, Faculty of Agriculture, Niigata University, Japan.
Hoshino
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D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D006094:Gram-Positive Bacteria; D006358:Hot Temperature; D019751:Intramolecular Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism*
D014364:Tryptophan; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase