Thesis (Ph.D.)--OGI School of Science & Engineering at OHSU, Oct. 2005. / Includes bibliographical references (leaves 115-134).

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Diane S. Herson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

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.

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,β-unsaturated-δ-lactams via a tandem C-H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a seven-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. With the goal of using protein engineering to improve the activity of monomeric streptavidin (mSav) ArM complexed to Rh(III), we also report computational calculations that demonstrate the effects of modifying the secondary coordination sphere. These findings have allowed us to engineer a more active mutant G49A that provides up to 67 more TON compared to WT mSav ArM. Directed evolution techniques, when applied to ArMs, allows us to mimic the natural selection process and thereby dramatically increase the TON of the ArM. This technology has been used to develop an artificial aminobrominase with up to 2587 TON.

Nitric oxide (NO) is a small, gaseous molecule that is toxic to life at high doses but serves a crucial role in biological processes at lower concentrations, including: cell signaling, immune response, and more recently, as a synthon in the biosynthesis of natural products in bacteria. Metalloenzymes are incredibly versatile catalysts that enable chemistry that often, still has no comparable laboratory reaction. TxtE, a cytochrome P450 (CYP), utilizes NO as a co-substrate along with dioxygen (O2) to catalyze the regioselective nitration of L-tryptophan (Trp) to produce 4-NO2-Trp. Work in this dissertation established that the TxtE ferric-superoxo intermediate is resistant to reduction, which facilitates its reaction with diffusible NO en route to an , as yet,-uncharacterized nitrating species. Furthermore, it is shown that an outer-sphere protein residue influences the nitration chemistry of TxtE. A Thr250Ala mutant version of TxtE characterized and found to lack all nitration ability despite maintaining cofactor incorporation and retaining competence for formation of the ferric-superoxo intermediate. Separately, experiments performed with wild-type TxtE demonstrate that analogs of Trp affect the lifetime of the ferric-superoxo intermediate and enable substrate hydroxylation. Additionally, a non-heme, diiron enzyme from Mycobacterium kansasii (MkaHLP) was previously established to possess NO peroxidase activity. In this dissertation, a Tyr54Phe mutant form of MkaHLP was characterized and found to have greatly diminished NO peroxidase activity due to the removal of the characteristic tyrosine ligand to the diiron site. Implications of this change in activity are discussed in the relevant section.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 152-164).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 180-187).

Thesis (M.S.)--Wake Forest University. Dept. of Chemistry, 2007. / Vita. Includes bibliographical references.