Ming Tien Professor of Biochemistry 408 Althouse Laboratory, University Park, PA 16802 Phone: (814) 863-1165 Fax: (814) 863-7024 Email: mxt3@psu.edu B.A. in anthropology, The University of Michigan Ph.D. biochemistry, Michigan State University Tien Lab Web Site

The Tien lab has three active research areas. Initial impression is that the research areas are not related; however, they share a common theme of redox reactions. These areas are summarized below.

Fungal lignin biodegradation

The degradation of lignin plays a key role in carbon recycling on earth. Lignin, an aromatic polymer, is second only to cellulose in abundances as a renewable carbon source and accounts for approximately 20% of all the carbons fixed by photosynthesis. Lignin is nature’s plastic imparting rigidity to woody biomass and conferring resistance to wood from most forms of microbial attack. The degradation of lignin is brought about predominantly by filamentous fungi. Due to the heterogeneity of woody biomass, an ensemble of enzymes is required to degrade this substrate to carbon dioxide. Both hydrolytic and oxidative enzymes are involved. The hydrolytic enzymes are used for depolymerization of cellulose whereas the oxidative enzymes are used for depolymerization of lignin. Our past research efforts has focused on the enzymology and regulation of the oxidative enzymes. These oxidative enzymes generate free radicals in lignin resulting in its depolymerization.

Despite years of research by many labs, the identity of the enzymes involved in wood degradation is still not known. Our past efforts have focused on mechanistic studies of peroxidases, Mn peroxidase and lignin peroxidase. However, to degrade wood, an ensemble of enzymes is required. The recent completion of the sequencing of a fungal genome, Phanerochaete chrysosporium and advances in methodology/instrumentation now allows us to identify all of the proteins produced to degrade wood. We are using a proteomics approach toward identifying all of the extracellular proteins produced by P. chrysosporium when grown on wood. Protein spots are excised from 2-dimensional gels, digested with trypsin and the peptides are sequenced by LC/MS/MS. Using this method, we are in the process of identifying the greater than 40 proteins produced when P. chrysosporium degrades oak. On going research involves determining the role of the wood substrate on enzyme production and the succession of enzymes involved in degradation of wood.

Dissimilatory Iron reduction

Micro-organisms are known to use over 20 elemental systems other than O2 to accept electrons during respiration. Of these, only 6 are known to be respired in solid form: S, As, Se, U, Fe, Mn. Of these six, the ability to reduce iron is the most common among deeply branching members of Archaea and Bacteria. Microbial Fe respiration is important because it dominates reduction of iron in a large number of natural systems today. Interest in this process, largely focused on Shewanella and Geobacter, has intensified recently because bioreduction of iron oxides releases co-precipitated or sorbed contaminants in the subsurface. Using the Shewanella oneidensis, we aim to elucidate the biochemical mechanism of iron reduction. Collaborating with Dr. Susan Brantley at Penn State’s Department of Geoscience, we have utilized a protemics approach toward understanding the enzymology of this process. Using 2-D gel electrophoresis, we have identified a number of proteins that are uniquely expressed under iron-reducing growth conditions. Because S. oneidensis has been recently sequenced, we have used trypsin fingerprinting using MALDI/TOF mass spectroscopy. In addition, we are the first team to develop an in vitro (cell-free) model system derived from Shewanella wherein membrane fractions directly reduce solid-phase Fe minerals. On going research aims to purify the enzymes involved in this process.

Methionine sulfoxide reductase

In collaboration with Dr. Eva Pell of Penn State University, we are characterizing the role of methionine sulfoxide reductases in protection of plants from oxidative stress. The generation of reactive oxygen species (ROS), especially under conditions of metabolic stress, is an unavoidable side effect of life in an oxygen atmosphere. To protect against ROS, biology has evolved both enzymatic and non-enzymatic scavenging systems. The enzymatic systems include superoxide dismutases, catalases and peroxidases. The non-enzymatic systems include antioxidants such as glutathione, vitamin E, carotene and vitamin C. ROS that circumvent the scavenging system can cause oxidative damage to virtually all biomolecules. Once damaged, certain macromolecules, such as DNA, are repaired by the cell. Research within the past decade has shown that proteins can also be repaired. Although all amino acids are susceptible to oxidative damage, Met residues, even more than Cys or Tyr, are the most susceptible. ROS readily oxidize Met residues by two-electrons to form the sulfoxide. The sulfoxide, in turn, can be reduced back to Met by the enzyme methionine sulfoxide reductase (PMSR). Arabidopsis has several copies of PMSR {Sadanandom, 2000 #256}. One of the gene products is targeted to the chloroplast whereas other three are believed to be cytosolic. We are utilizing transgenic plants, ones that over express and under express these enzymes to determine their role in oxidative stress. We are also charactering the enzymology of these enzymes.

Representative Publications:

• Ambert-Balay, K., Dougherty, M., Tien, M. (2000) Reactivity of manganese peroxidase: site-directed mutagenesis of residues in proximity to the porphyrin ring. Arch. Biochem. Biophys. 382, 89-94.
• Mester, T., K. Ambert-Balay, et al. (2001). "Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase." J. Biol. Chem. 276(25): 22985-22990.
• Mester, T. and M. Tien (2001). "Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium." Biochem. Biophys. Res. Commun. 284(3): 723-728.
• Mester, T. and M. Tien (2001). "Oxidation mechanism of Ligninolytic Enzymes Involved in the Degradation of Environmental Pollutants." Intern. Biodet. Biodeg. 46: 51-59.
• Banci, L., Bartalesi, I., Ciofi-Baffoni, S. and Tien, M. (2003) “Unfolding and pH studies on manganese peroxidase: Role of heme and calcium on secondary structure stability” Biopolymers 72: 38-47.
• Varela, E. and M. Tien, Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation. Appl. Environ. Microbiol, 2003. 69(10): p. 6025-6031.
• Varela, E., T. Mester, and M. Tien, Culture conditions affecting biodegradation components of the brown-rot fungus Gloeophyllum trabeum. Arch. Microbiol., 2003. 180(4): p. 251-256.
• Icopini, G.A., A.D. Anbar, S.S. Ruebush, M. Tien, and S.L. Brantley, Iron isotope fractionation during microbial reduction of iron: The importance of adsorption. Geology, 2004. 32: p. 205–208.
• Ruebush, S. S.; Icopini, G. A.; Brantley, S. L.; Tien, M. “In Vitro Enzymatic Mineral Oxide Reduction by Membrane Fractions from Shewanella oneidensis MR1” Enzyme Microb. Technol. 2004, submitted.
• Romero, H. M., Jensen, P., Berlett, B, Pell, E. and Tien, M. (2004) “The Plastidial Peptide Methionine Sulfoxide Reductase is an Important Component of the Response of Arabidopsis to Oxidative Stress in the Chloroplast” Plant Phys. Submitted.