Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. The maturation of MADH involves at least 4 other proteins, and we have begun to characterize one of these, MauG. It is a highly unusual di-heme enzyme responsible for the completion of TTQ synthesis. The natural substrate for MauG (preMADH) is a 119-kDa protein precursor of MADH with a partially formed cofactor. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis, using three moles of either molecular oxygen or hydrogen peroxide as the second substrate. The two hemes of MauG act as a single redox unit, and the catalytic reaction involves an unprecedented high-valent di-heme intermediate that is unusually stable. The high-valent species is an FeV equivalent consisting of one FeIV=O heme, with the other in an FeIV oxidation state. During the previous funding period we have solved the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1 ?, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The MauG-preMADH structure, along with other recent data from collaborating labs, has been full of surprises. The structure indicates there are no major structural rearrangements during catalysis and that long-range inter-protein electron and radical transfer occurs in this system. During catalysis a very stable MADH protein radical is also produced. Now we wish to move the crystallographic studies, complemented by mass spectrometry and single crystal spectroscopies, beyond what was envisioned during the previous funding period, and make specific discoveries about oxygen activation by a previously unknown high-valent iron intermediate, mechanisms of oxidative modification to specific amino acid residues within a protein, and stabilization of highly reactive oxygen species (ROS) and radicals. Fundamentally these studies will give molecular insight into inter-protein electron transfer central to metabolis, and the control and outcomes of oxidative damage by radicals and ROS that are associated with many disease states. Thus protective control of electrons, radicals and ROS within the protein matrix is a key component of human health.

Public Health Relevance

MauG is sequentially related to peroxidases that detoxify H2O2 under conditions of oxidative stress, but unusually can also activate molecular oxygen, forming a highly reactive iron oxidant that is unusually stable. In other systems such species are transient, and lead to non-specific oxidative tissue damage associated with many disease states if allowed to linger. How can MauG control such a potentially damaging species?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM066569-10A1
Application #
8367992
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2002-07-01
Project End
2016-07-31
Budget Start
2012-09-01
Budget End
2013-07-31
Support Year
10
Fiscal Year
2012
Total Cost
$292,089
Indirect Cost
$92,089
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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
Klema, Valerie J; Solheid, Corinne J; Klinman, Judith P et al. (2013) Structural analysis of aliphatic versus aromatic substrate specificity in a copper amine oxidase from Hansenula polymorpha. Biochemistry 52:2291-301
Abu Tarboush, Nafez; Jensen, Lyndal M R; Wilmot, Carrie M et al. (2013) A Trp199Glu MauG variant reveals a role for Trp199 interactions with pre-methylamine dehydrogenase during tryptophan tryptophylquinone biosynthesis. FEBS Lett 587:1736-41
Wilmot, Carrie M; Yukl, Erik T (2013) MauG: a di-heme enzyme required for methylamine dehydrogenase maturation. Dalton Trans 42:3127-35
Davidson, Victor L; Wilmot, Carrie M (2013) Posttranslational biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Annu Rev Biochem 82:531-50
Makris, Thomas M; Knoot, Cory J; Wilmot, Carrie M et al. (2013) Structure of a dinuclear iron cluster-containing *-hydroxylase active in antibiotic biosynthesis. Biochemistry 52:6662-71
Abu Tarboush, Nafez; Yukl, Erik T; Shin, Sooim et al. (2013) Carboxyl group of Glu113 is required for stabilization of the diferrous and bis-Fe(IV) states of MauG. Biochemistry 52:6358-67
Yukl, Erik T; Liu, Fange; Krzystek, J et al. (2013) Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis. Proc Natl Acad Sci U S A 110:4569-73
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

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