From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase

被引:144
作者
Lehmann, M [1 ]
Kostrewa, D [1 ]
Wyss, M [1 ]
Brugger, R [1 ]
D'Arcy, A [1 ]
Pasamontes, L [1 ]
van Loon, APGM [1 ]
机构
[1] F Hoffmann La Roche & Co Ltd, CH-4070 Basel, Switzerland
来源
PROTEIN ENGINEERING | 2000年 / 13卷 / 01期
关键词
animal feed pelleting; consensus protein design; increased heat stability; phytase family; three-dimensional structure;
D O I
10.1093/protein/13.1.49
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase, In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.
引用
收藏
页码:49 / 57
页数:9
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