Found in all kingdoms of life, copper-containing amine oxidases (CuAOs) contain two redox centers. One is a copper ion, and the other is a novel organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), derived from the post-translational modification of a Tyr residue in the protein. The biosynthesis of TPQ is an auto-catalytic process, which is molecular oxygen and cupric ion dependent. Formation of such self- processed cofactors likely played a key role in enzyme evolution, as they extended the chemical functionalities available for catalysis without the evolution of separate biosynthetic enzymes. Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, also contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. In contrast to CuAOs, the maturation of MADH involves at least 4 other proteins. In the previous grant period, we have begun to characterize one of these proteins, MauG. It is a highly unusual diheme enzyme responsible for the completion of TTQ synthesis, which can use either molecular oxygen or hydrogen peroxide as a substrate. In the last few years, the list of enzymes containing amino acid derived cofactors has grown rapidly. In addition, both TPQ and TTQ synthesis require oxygen substrates, but in very different reactions. The ability to activate molecular oxygen underpins all aerobic biology, but the details of how this is achieved are still poorly understood. I propose to use the yeast Hansenula polymorpha methylamine oxidase (HPAO) to study the structural basis of TPQ formation. Through a novel combination of single crystal UV/visible microspectrophotometry, X-ray crystallography and anaerobic / aerobic freeze trapping, reaction intermediates in biogenesis will be trapped in the crystal. The controversial role of copper in CuAO will be explored through metal replacement studies. In addition, I propose to explore MADH maturation through X-ray crystallographic studies of MauG, in particular focusing on the atypical behavior of the c-type heme(s).

Public Health Relevance

The aberrant actions of human copper-containing amine oxidases are linked to congestive heart disease, late-diabetic complications and Alzheimer's disease. One human copper-containing amine oxidase is involved in the inflammatory response, and represents a new target for anti-inflammatory drugs. MauG is sequentially related to peroxidases that detoxify H2O2 under hypoxic conditions (oxidative stress), but unusually can also activate molecular oxygen with mechanistic similarities to human cytochrome P450 enzymes, the major players in drug metabolism, carcinogen activation, biosynthesis of physiologically important molecules, such as steroids, fat-soluble vitamins and fatty acids, as well as the degradation of insecticides and herbicides.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066569-07
Application #
7587378
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2002-07-01
Project End
2012-01-31
Budget Start
2009-02-01
Budget End
2010-01-31
Support Year
7
Fiscal Year
2009
Total Cost
$277,214
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Barr, Ian; Stich, Troy A; Gizzi, Anthony S et al. (2018) X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE. Biochemistry 57:1306-1315
Tu, Xiongying; Latham, John A; Klema, Valerie J et al. (2017) Crystal structures reveal metal-binding plasticity at the metallo-?-lactamase active site of PqqB from Pseudomonas putida. J Biol Inorg Chem 22:1089-1097
Evans 3rd, Robert L; Latham, John A; Xia, Youlin et al. (2017) Nuclear Magnetic Resonance Structure and Binding Studies of PqqD, a Chaperone Required in the Biosynthesis of the Bacterial Dehydrogenase Cofactor Pyrroloquinoline Quinone. Biochemistry 56:2735-2746
Evans 3rd, Robert L; Latham, John A; Klinman, Judith P et al. (2016) (1)H, (13)C, and (15)N resonance assignments and secondary structure information for Methylobacterium extorquens PqqD and the complex of PqqD with PqqA. Biomol NMR Assign 10:385-9
Roessler, Christian G; Agarwal, Rakhi; Allaire, Marc et al. (2016) Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography. Structure 24:631-640
Shin, Sooim; Feng, Manliang; Li, Chao et al. (2015) A T67A mutation in the proximal pocket of the high-spin heme of MauG stabilizes formation of a mixed-valent FeII/FeIII state and enhances charge resonance stabilization of the bis-FeIV state. Biochim Biophys Acta 1847:709-16
Shin, Sooim; Yukl, Erik T; Sehanobish, Esha et al. (2014) Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG. Biochemistry 53:1342-9
Cheng, Zhongjun; Cheung, Peggie; Kuo, Alex J et al. (2014) A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes Dev 28:1758-71
Yukl, Erik T; Williamson, Heather R; Higgins, LeeAnn et al. (2013) Oxidative damage in MauG: implications for the control of high-valent iron species and radical propagation pathways. Biochemistry 52:9447-55
Johnson, Bryan J; Yukl, Erik T; Klema, Valerie J et al. (2013) Structural snapshots from the oxidative half-reaction of a copper amine oxidase: implications for O2 activation. J Biol Chem 288:28409-17

Showing the most recent 10 out of 49 publications