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

Morgan Desjardins, Wai Shun Mak, Terrence E. O'Brien, Dylan Alexander Carlin, Dean J. Tantillo, Justin Siegel

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 languageEnglish (US)
Pages (from-to)3308-3314
Number of pages7
JournalACS Omega
Volume2
Issue number7
DOIs
StatePublished - Jul 31 2017

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L-threonine 3-dehydrogenase
Functional analysis
Enzymes
Substrates
Catalysis
Enzyme activity
Hydrides
Oxidoreductases
Proteins

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 journalArticle

Desjardins, Morgan ; Mak, Wai Shun ; O'Brien, Terrence E. ; Carlin, Dylan Alexander ; Tantillo, Dean J. ; Siegel, Justin. / Systematic Functional Analysis of Active-Site Residues in l -Threonine Dehydrogenase from Thermoplasma volcanium. In: ACS Omega. 2017 ; Vol. 2, No. 7. pp. 3308-3314.
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