Embargo End Date2016-12-09
Permanent link to this recordhttp://hdl.handle.net/10754/583809
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Access RestrictionsAt the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation became available to the public after the expiration of the embargo on 2016-12-09.
AbstractThe 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.
CitationFischer, J. (2015). A Pathway to Artificial Metalloenzymes. KAUST Research Repository. https://doi.org/10.25781/KAUST-HC787