Systematic Functional Analysis of Active-Site Residues in l -Threonine Dehydrogenase from Thermoplasma volcanium
[1];
[1];
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States, Department of Biochemistry and Molecular Medicine, University of California, Davis, 2700 Stockton Boulevard, Suite 2102, Sacramento, California 95817, United States, Genome Center, University of California, Davis, 451 Health Sciences Drive, Davis, California 95616, United States
Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs shows that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.
- Research Organization:
- Univ. of California at Davis, Davis, CA (United States)
- Sponsoring Organization:
- USDOE Advanced Research Projects Agency - Energy (ARPA-E)
- Grant/Contract Number:
- AR0000429
- OSTI ID:
- 1368598
- Alternate ID(s):
- OSTI ID: 1415020
- Journal Information:
- ACS Omega, Journal Name: ACS Omega Vol. 2 Journal Issue: 7; ISSN 2470-1343
- Publisher:
- American Chemical SocietyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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