The advancement of catalytic systems and the application thereof has proven to be the key to
overcome traditional limitations of industrial-scale synthetic processes. Converging
organometallic and biocatalytic principles lead to the development of Artificial Metalloenzymes
(ArMs) that comprise a synthetic metal catalyst embedded in a protein scaffold, thereby
combining the reactivity of the former with the versatility of the latter. This synergistic
approach introduces rationally designed building blocks for the catalytic site and the host protein
to assemble enzyme-like structures that follow regio-, chemo-, enantio- and substrate-selective
principles. Yet, the identification of suitable protein scaffolds has thus far been challenging.
Herein we report a rationally optimized fluorescent protein host, mTFP*, that was engineered to
have no intrinsic metal binding capability and, owing to its robust nature, can act as scaffold for
the design of novel ArMs. We demonstrate the potential of site-specific modifications within the
protein host, use protein X-Ray analysis to validate the respective scaffolds and show how
artificial mutant binding sites can be introduced. Transition metal Förster Resonance Energy
transfer (tmFRET) methodologies help to evaluate micromolar dissociation constants and reveal
structural rearrangements upon coordination of the metal centers. In conjunction with molecular
insights from X-Ray crystallographic structure determination, dynamics of the binding pocket can
be inferred. The versatile subset of different binding motifs paired with transition metal catalysts
create artificial metalloenzymes that provide reactivities which otherwise do not exist in nature.
As a proof of concept, Diels-Alder cycloadditions highlight the potential of the present mTFP*
based catalysts by stereoselectively converting azachalcone and cyclopentadiene substrates.
Screens indicate an enantiomeric excess of up to 60% and provide insights into the electronic and
geometric constitution of the first coordination spheres binding the catalysts.
We further apply two general principles to optimize selective conversions of the generated ArMs.
1) Utilizing site-specific mutagenesis, increased hydrophobicity is introduced to the second coordination sphere. 2) In-vitro post-expressional modification utilizing N-hydroxysuccinimide
esters is anticipated to introduce a sterically more demanding second coordination sphere that
influences substrate entry by favoring a particular stereoisomer. The latter approach however also
enhances the host proteins robustness under processing conditions.
The presented study investigates a novel approach to create artificial metalloenzymes based on
non-enzymatic precursor proteins. It illustrates means of modification and functionalization.
Further guidance to overcome the general problem of insufficient stereoselectivity and stability is
also presented. In view of the insights gained we see the importance of further mutagenic studies,
i.e. through means of guided evolution, to extend stereoselectivities. In-vivo applications of
artificial metalloenzymes could thus be used to pursue metabolomic engineering.
|Date of Award||Dec 2015|
|Original language||English (US)|
- Biological, Environmental Science and Engineering
|Supervisor||Jorg Eppinger (Supervisor)|
- fluorescent protein