From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase.
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.
Lehmann
M
M
F.Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland. Martin.Lehmann@Roche.com
Kostrewa
D
D
Wyss
M
M
Brugger
R
R
D'Arcy
A
A
Pasamontes
L
L
van Loon
A P
AP
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D011994:Recombinant Proteins; D010832:6-Phytase