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. These results
are directly applicable to rational enzyme design and engineering.