Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a family of
enzymes that
play important roles in regulating cellular signaling pathways. The
activity of these enzymes is regulated by the motion of a catalytic
loop that places a critical conserved aspartic acid side chain into
the active site for acid–base catalysis upon loop closure.
These enzymes also have a conserved phosphate-binding loop that is
typically highly rigid and forms a well-defined anion-binding nest.
The intimate links between loop dynamics and chemistry in these enzymes
make PTPs an excellent model system for understanding the role of
loop dynamics in protein function and evolution. In this context,
archaeal PTPs, which have often evolved in extremophilic organisms,
are highly understudied, despite their unusual biophysical properties.
We present here an engineered chimeric PTP (ShufPTP) generated by
shuffling the amino acid sequence of five extant hyperthermophilic
archaeal PTPs. Despite ShufPTP’s high sequence similarity to
its natural counterparts, it presents a suite of unique properties,
including high flexibility of the phosphate binding P-loop, facile
oxidation of the active-site cysteine, mechanistic promiscuity, and,
most notably, hyperthermostability, with a denaturation temperature
likely >130 °C (>8 °C higher than the highest recorded
growth
temperature of any archaeal strain). Our combined structural, biochemical,
biophysical, and computational analysis provides insight both into
how small steps in evolutionary space can radically modulate the biophysical
properties of an enzyme and showcases the tremendous potential of
archaeal enzymes for biotechnology, to generate novel enzymes capable
of operating under extreme conditions.