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Reengineering substrate specificity of E. coli glutamate dehydrogenase using a position-based prediction method
Feng Geng1, Cheng-Wei Ma1, An-Ping Zeng2
1Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21073, Hamburg, Germany.
Objective:
To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).
Results:
The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg-1.
Conclusion:
The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.
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