|
Stephen A. Mills, Ph.D.
Department of Chemistry and Biochemistry Office: ST 485 Phone: (619) 260-7564 Email: smills@sandiego.edu
|
 |
B.S. University of Illinois at Urbana-Champaign
M.S. University of California, Berkeley, Advisor: Paul A. Bartlett
Ph.D. University of California, Berkeley, Advisor: Judith P. Klinman
Post-doctoral Fellow, University of California, Berkeley, Advisor: Michael A. Marletta
Courses Taught
Spring 2007
Chemistry 302 Organic Chemistry II syllabus
Chemistry 335 Biochemistry Lab syllabus
Fall 2006
Chemistry 301 Organic Chemistry syllabus
Research
Interests
The theme of research in our lab is to understand the role of metals in proteins. We are interested in how specific metals get into proteins and why the specific metals are important for the function of the protein. A primary technique used in our lab is metal substitution. We then use a variety of techniques to evaluate the effect of different metals on the function of the protein, including enzyme kinetics, gel-shift assays, UV-vis and fluorescence spectroscopy.
Specific Systems under study:
Ferric
Uptake Regulator –
Fur
Selected References
Fur is a key part of the bacterial iron metabolism. Most organisms require iron for survival, however the predominant form, Fe3+, is very insoluble under physiological conditions. Bacteria expend considerable energy scavenging iron from their environment to support their growth. Although required for growth, too much iron can be toxic. Fur is an iron-sensing protein in bacteria. When there is sufficient iron in the cell, iron binds to Fur and activates the protein to bind DNA. Upon binding DNA, Fur turns off expression of iron importing proteins and prevents the buildup of toxic iron levels. If metals, other than iron, activate Fur for DNA binding, iron import can be turned off when it should remain on and starve the cell for iron. We are examining the affinity and selectivity of Fur for various metals, including Mn, Fe, Co, and Zn to understand how selective the protein needs to be for proper bacterial growth. By examining Fur homologues, with known primary sequence, from various bacterial species we hope to identify specific amino acids that are important for metal selectivity. Site-directed mutagenesis will be used to alter the metal selectivity in specific homologues.
Copper
Amine Oxidase –
CAO
Selected References
The copper amine oxidases (CAOs) are a family of enzymes that oxidize amines using a unique, protein-bound cofactor, trihydroxyphenylalanine quinone (TPQ), and a Cu2+ ion. The cupric ion in these proteins is important for formation of the cofactor and for catalysis once the cofactor is in place. CAOs are functional as dimers with one TPQ and one Cu2+ in each monomer. We are interested in the ability of the active site of one monomer to interact with the active site of its partner, especially how the metal in one site affects the activity of the other active site.
CoaR
– A cobalt-specific
regulatory protein
Selected References
This is the only known regulatory protein specific for Co in bacteria. It is a part of the vitamin B12 biosynthetic pathway. Little is known about this protein except that it responds to Co and a B12 precursor – hydrogenobyrinic acid. We want to examine its selectivity for cobalt over other first-row transition metals and explore the effect of hydrogenobyrinic acid on metal affinity.
McHugh, J.P., et al., Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J Biol Chem, 2003. 278(32): p. 29478-86.
Mills, S.A. and M.A. Marletta, Metal binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli. Biochemistry, 2005. 44(41): p. 13553-9.
Pohl, E., et al., Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol Microbiol, 2003. 47(4): p. 903-15.
Mills, S.A., et al., Mechanistic comparison of the cobalt-substituted and wild-type copper amine oxidase from Hansenula polymorpha. Biochemistry, 2002. 41(34): p. 10577-84.
Mure, M., S.A. Mills, and J.P. Klinman, Catalytic mechanism of the topa quinone containing copper amine oxidases. Biochemistry, 2002. 41(30): p. 9269-78.
Rutherford, J.C., J.S. Cavet, and N.J. Robinson, Cobalt-dependent transcriptional switching by a dual-effector MerR-like protein regulates a cobalt-exporting variant CPx-type ATPase. J Biol Chem, 1999. 274(36): p. 25827-32.
Scott, A.I., Discovering nature's diverse pathways to vitamin B12: a 35-year odyssey. J Org Chem, 2003. 68(7): p. 2529-39.
Tottey, S., D.R. Harvie, and N.J. Robinson, Understanding how cells allocate metals using metal sensors and metallochaperones. Acc Chem Res, 2005. 38(10): p. 775-83.