Abstract
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. These results are directly applicable to rational enzyme design and engineering.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3308-3314 |
| Number of pages | 7 |
| Journal | ACS Omega |
| Volume | 2 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 31 2017 |
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ASJC Scopus subject areas
- Chemical Engineering(all)
- Chemistry(all)
Cite this
Systematic Functional Analysis of Active-Site Residues in l -Threonine Dehydrogenase from Thermoplasma volcanium. / Desjardins, Morgan; Mak, Wai Shun; O'Brien, Terrence E.; Carlin, Dylan Alexander; Tantillo, Dean J.; Siegel, Justin.
In: ACS Omega, Vol. 2, No. 7, 31.07.2017, p. 3308-3314.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Systematic Functional Analysis of Active-Site Residues in l -Threonine Dehydrogenase from Thermoplasma volcanium
AU - Desjardins, Morgan
AU - Mak, Wai Shun
AU - O'Brien, Terrence E.
AU - Carlin, Dylan Alexander
AU - Tantillo, Dean J.
AU - Siegel, Justin
PY - 2017/7/31
Y1 - 2017/7/31
N2 - 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. These results are directly applicable to rational enzyme design and engineering.
AB - 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. These results are directly applicable to rational enzyme design and engineering.
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U2 - 10.1021/acsomega.7b00519
DO - 10.1021/acsomega.7b00519
M3 - Article
AN - SCOPUS:85028927060
VL - 2
SP - 3308
EP - 3314
JO - ACS Omega
JF - ACS Omega
SN - 2470-1343
IS - 7
ER -