ConspectusThe biotechnological revolution has made it
possible to create
enzymes for many reactions by directed evolution. However, because
of the immense number of possibilities, the availability of enzymes
that possess a basal level of the desired catalytic activity is a
prerequisite for success. For new-to-nature reactions, artificial
metalloenzymes (ARMs), which are rationally designed hybrids of proteins
and catalytically active transition-metal complexes, can be such a
starting point.This Account details our efforts toward the
creation of ARMs for
the catalysis of new-to-nature reactions. Key to our approach is the
notion that the binding of substrates, that is, effective molarity,
is a key component to achieving large accelerations in catalysis.
For this reason, our designs are based on the multidrug resistance
regulator LmrR, a dimeric transcription factor with a large, hydrophobic
binding pocket at its dimer interface. In this pocket, there are two
tryptophan moieties, which are important for promiscuous binding of
planar hydrophobic conjugated compounds by π-stacking. The catalytic
machinery is introduced either by the covalent linkage of a catalytically
active metal complex or via the ligand or supramolecular assembly,
taking advantage of the two central tryptophan moieties for noncovalent
binding of transition-metal complexes.Designs based on the
chemical modification of LmrR were successful
in catalysis, but this approach proved too laborious to be practical.
Therefore, expanded genetic code methodologies were used to introduce
metal binding unnatural amino acids during LmrR biosynthesis in vivo.
These ARMs have been successfully applied in Cu(II) catalyzed Friedel–Crafts
alkylation of indoles. The extension to MDRs from the TetR family
resulted in ARMs capable of providing the opposite enantiomer of the
Friedel–Crafts product. We have employed a computationally
assisted redesign of these ARMs to create a more active and selective
artificial hydratase, introducing a glutamate as a general base at
a judicious position so it can activate and direct the incoming water
nucleophile.A supramolecularly assembled ARM from LmrR and
copper(II)–phenanthroline
was successful in Friedel–Crafts alkylation reactions, giving
rise to up to 94% ee. Also, hemin was bound, resulting in an artificial
heme enzyme for enantioselective cyclopropanation reactions. The importance
of structural dynamics of LmrR was suggested by computational studies,
which showed that the pore can open up to allow access of substrates
to the catalytic iron center, which, according to the crystal structure,
is deeply buried inside the protein.Finally, the assembly approaches
were combined to introduce both
a catalytic and a regulatory domain, resulting in an ARM that was
specifically activated in the presence of Fe(II) salts but not Zn(II)
salts.Our work demonstrates that LmrR is a privileged scaffold
for ARM
design: It allows for multiple assembly methods and even combinations
of these, it can be applied in a variety of different catalytic reac...