Copper-containing enzymes exhibit remarkable diversity in both structure and function, and their copper active sites play major roles in biological dioxygen activation. The selective O2 activation in polysaccharide monooxygenases (PMOs) and the family of non-coupled binuclear copper enzymes is well-suited for biological functions that require activation of oxygen and generation of reactive Cu/O2 species under specific conditions. If not carefully regulated, promiscuous reactive oxygen species can react non-specifically with essential biomolecules once O2 is activated, leading to maladies such as Alzheimer disease. The proposed work aims to understand the mechanisms of O2 activation and the stereo- and regio-specific interactions of proposed Cu/O2 reactive species with substrates in auxiliary activity 9 (AA9)-PMOs and the non-coupled binuclear copper enzymes peptidyl ?-hydroxylating monooxygenase (PHM), dopamine ?-Monooxygenase (D?M), and tyramine ?-Monooxygenase (T?M). AA9-PMOs cleave crystalline cellulose, are of considerable importance in the developing area of second-generation cellulosic biofuels, and have recently been associated with meningitis and intravenous infection in mouse models. The non-coupled binuclear copper enzymes PHM and D?M/T?M also carry out important biological and neurochemical functions ranging from catalyzing the first step in amidation of neuropeptide hormones required for normal developmental transitions to hydroxylation of the ?- carbon of aminoethyl substituted hormone substrates in processes vital to the regulation of neurotransmitters, respectively. Understanding the functions of these enzymes and exploiting them in various applications, either in development of alternative biofuels or in treatment of diseases, requires a thorough understanding of their copper centers and their proposed Cu/O2 intermediates. Previous studies by the Solomon lab and others have contributed to the prevailing hypotheses for the mechanisms of O2 activation, H-atom abstraction, and regulation in these enzymes. The proposed studies are designed to test these mechanisms at the molecular level, using spectroscopic techniques in combination with theoretical calculations to characterize the electronic and geometric structures of key intermediates in the catalytic cycles of AA9-PMO, PHM, and T?M. We propose methods to tune the catalytic cycles (site-directed mutagenesis studies, use of `slow' substrates) to trap putative intermediates, and will use rapid freeze quench techniques to prepare samples for study by X-ray absorption, rRaman, EPR, MCD, and K? X-ray emission spectroscopies. Computational methods will be used in combination with these spectroscopic techniques to extract further information on the frontier molecular orbitals and reaction coordinates. It is our goal to provide complete spectroscopic characterizations of the intermediates involved in O2 activation and H-atom abstraction in these enzymes.
Copper-dependent monooxygenase enzymes play important roles in biological and neurological health issues and their malfunctions have been linked to Alzheimer's disease, certain cancers, and infection. The proposed work aims to understand the mechanisms of O2 activation and H-atom abstraction reactivity in polysaccharide monooxygenases and the family of non-coupled binuclear copper enzymes, focusing on the stereo- and regio-specific interactions of their proposed Cu/O2 intermediates with substrates that prevent formation of promiscuous reactive oxygen species. Utilizing a combination of spectroscopic and computational techniques we will explore the mechanisms of these enzymes at the molecular level and characterize the electronic and geometric structures of key intermediates in an effort to understand and address these issues.
| Meier, Katlyn K; Jones, Stephen M; Kaper, Thijs et al. (2018) Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars. Chem Rev 118:2593-2635 |
| Hansson, Henrik; Karkehabadi, Saeid; Mikkelsen, Nils et al. (2017) High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain. J Biol Chem 292:19099-19109 |
