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

Citation
M. Lehmann et al., From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase, PROTEIN ENG, 13(1), 2000, pp. 49-57
Citations number
43
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN ENGINEERING
ISSN journal
0269-2139 → ACNP
Volume
13
Issue
1
Year of publication
2000
Pages
49 - 57
Database
ISI
SICI code
0269-2139(200001)13:1<49:FDSTIF>2.0.ZU;2-M
Abstract
Naturally-occurring phytases having the required level of thermostability f or application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequ ence comparisons. A consensus enzyme based on 13 fungal phytase sequences h ad normal catalytic properties, but showed an unexpected 15-22 degrees C in crease in unfolding temperature compared with each of its parents. As a fir st step towards understanding the molecular basis of increased heat resista nce, 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 e xpected, based on comparisons of both three-dimensional structures, to cont ribute more to phytase stabilization than non-consensus amino acids. For so me consensus amino acids, predicted by structural comparisons to destabiliz e the protein, mutational analysis was performed. Interestingly, these cons ensus residues in fact increased the unfolding temperature of the consensus phytase, In summary, for fungal phytases apparently an unexpected direct l ink between protein sequence conservation and protein stability exists.