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Contribution of the ion pairR60-D27* to the properties of thermophilic xylose isomerase determined by site-directed mutagenesis

Wei Xu, Rong Shao, Ming Yan, Pingkai Ouyang

The role of ion pair R60-D27* on the subunit interface of Thermus thermophilus xylose isomerase was investigated by site-directed mutagenesis and structure analysis. The conserved residue Arg60 in thermophilic xylose isomerases was substituted with hydrophobic residue (Phe) in the mesophilic counterparts. The results show that the maximal specific activity of themutantV144A/R60F is 32.1 U/mg at 85 °C, which is about 10.46% of V144A, and exhibits an obvious decline in alkaline environment.As to thermostability, after 4 h incubation at 75 °C, the V144A displays about 80% of its initial activity while the mutant V144A/R60F retains only about 50%. Moreover, the catalytic efficiency of mutant V144A/ R60F is only 25.34%of that of V144A, which shows an obvious decrease. The structure comparison of V144A and V144A/R60F reveals that the residue D27 shifts slightly outward because of the disruption of ion pair R60-D27*. This affects indirectly the conserved residue F25which belongs to the active site. The above analysis indicates that the special ion pair R60-D27* plays an important role in keeping the high thermostability, and whatÂ’s more, it helps to maintain the proper conformation of F25* in the active site of thermophilic xylose isomerases.