Date of Award
Summer 2019
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Holz, Richard C.
Second Advisor
Fiedler, Adam
Third Advisor
kincaid, James R.
Abstract
Metalloenzymes are widely involved in water incorporation across CN and C-Cl bonds.1 In this research, the structure and mechanisms of two novel metalloenzymes that incorporate water across CN and C-Cl bonds were examined by biophysical approaches, computational modeling, and crystallographic methods.In the research of water incorporation across CN bonds, a novel eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase) was characterized, and the functional role of a (His)17 section and an insert region in the MbNHase, were examined by gene modifications. Each of these MbNHase enzymes provided an 22 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions The kinetic studies and metal analysis establish that neither the (His)17 nor the entire insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.In the research of water incorporation across C-Cl bonds, a novel Zn(II)-dependent chlorothalonil hydrolytic dehalogenase from Pseudomonas sp. CTN-3 (Chd) was characterized and analyzed by a new direct spectrophotometric assay, metal analysis, and crystallography methods. A single Zn(II) ions was found to bind per Chd monomer. Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0 while multiple turnover pre-steady state stopped-flow data suggested a three-step model. The combination of these data along with pH dependent studies, allowed a catalytic mechanism for Chd to be proposed for the first time. The X-ray crystal structure of a fifteen residue N-terminal truncated form of the Chd (ChdT) was solved using single wavelength anomalous dispersion (SAD) to a resolution of 1.96 Å in the primitive orthorhombic space group P212121. ChdT is the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT is a “head-to-tail” homodimer, formed between two -helices from each monomer, with three Zn(II) binding sites, one of which is the active site Zn(II) while the third forms a structural site at the homodimer interface. The structural Zn(II) ion is not accessible to the bulk solvent. The active site Zn(II) ion resides in a slightly distorted trigonal bipyramid or TBP geometry with His117, His257, Asp116, Asn216, and water/hydroxide as ligands. A conserved His residue (His114) is hydrogen bound to the Zn(II) bound water/hydroxide and likely functions as the general acid/base. Substrate binding was examined by docking TPN into the hydrophobic channel with the most energetically favorable pose occurring for a TPN orientation that coordinates to the active site Zn(II) ions via a CN and that maximizes a π-π interaction with Trp227. The combination of the structure and substrate docking studies with the previously reported kinetic studies, has allowed a refined catalytic mechanism to be propose.