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Title: Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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.
Authors:
ORCiD logo [1] ;  [1] ;  [1] ;  [1] ; ORCiD logo [1] ;  [1]
  1. Univ. of California, Davis, CA (United States)
Publication Date:
Grant/Contract Number:
AR0000429
Type:
Published Article
Journal Name:
ACS Omega
Additional Journal Information:
Journal Volume: 2; Journal Issue: 7; Journal ID: ISSN 2470-1343
Publisher:
American Chemical Society (ACS)
Research Org:
Univ. of California at Davis, Davis, CA (United States)
Sponsoring Org:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Enzyme kinetics; Proteins; Quantum mechanics
OSTI Identifier:
1368598
Alternate Identifier(s):
OSTI ID: 1415020

Desjardins, Morgan, Mak, Wai Shun, O’Brien, Terrence E., Carlin, Dylan Alexander, Tantillo, Dean J., and Siegel, Justin B.. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium. United States: N. p., Web. doi:10.1021/acsomega.7b00519.
Desjardins, Morgan, Mak, Wai Shun, O’Brien, Terrence E., Carlin, Dylan Alexander, Tantillo, Dean J., & Siegel, Justin B.. Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium. United States. doi:10.1021/acsomega.7b00519.
Desjardins, Morgan, Mak, Wai Shun, O’Brien, Terrence E., Carlin, Dylan Alexander, Tantillo, Dean J., and Siegel, Justin B.. 2017. "Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium". United States. doi:10.1021/acsomega.7b00519.
@article{osti_1368598,
title = {Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium},
author = {Desjardins, Morgan and Mak, Wai Shun and O’Brien, Terrence E. and Carlin, Dylan Alexander and Tantillo, Dean J. and Siegel, Justin B.},
abstractNote = {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.},
doi = {10.1021/acsomega.7b00519},
journal = {ACS Omega},
number = 7,
volume = 2,
place = {United States},
year = {2017},
month = {7}
}