Dear CCP4bb,
We work on an enzyme that we crystallized with two substrates bound in
the active site (the reaction transform two substrates into two
products). We have also the structure with the two products. We are
able to see densities for the substrates when we collect data at
different time point post-crystallization (days or weeks later). There
is no change over time and no in crystallo enzymatic reaction despite
the fact that in solution using the same crystallization solution, the
reaction occurs readily.
This is not surprising and there are already many examples in the literature.
However, when we crystallize a single amino acid variant (mutant
within the active site) with the same two substrates, we initially see
the substrates but we then observe in crystallo enzymatic activity and
formation of the final products over time. This structure is identical
to the one determined with the two products co-crystallized with the
enzyme. The crystal packing does not seem to be at play here.
I understand that in crystallo activities are well documented in the
literature and can be induced by addition of ligands, X-rays, change
in oxidative environment, etc?
Here, the substrates are present from the beginning of the
crystallization experiments with the same concentration. Nothing is
added to the crystals later on. Only the time differentiate the two
type of crystals: after a couple of weeks, one has the substrates in
the active site (wt) while the other has the products (variant).
Is anyone aware of similar examples where a variant induce in
crystallo enzymatic activity without perturbation of the crystal?
Thanks,
Pierre
Dept of Pharmacology, Toxicology and cellular signaling
Paris Descartes University
