B.S. Chemistry, University of Washington, Seattle, WA
Ph.D. Biological Chemistry, M.I.T, Cambridge, MA
Solving Interesting Problems in Enzymology
I am an enzymologist who solves interesting and difficult questions related to the mechanism and function of enzymes towards a better understanding of biological processes. Over the past few years I have been active in the Copley lab where I have worked on the biosynthetic pathway for pyridoxal phosphate (PLP) and on the degradation pathway of pentachlorophenols (PCPs).
In PLP biosynthesis my work has focused on the enzyme PdxB, which catalyzes the second step in the biosynthetic pathway for PLP in E. coli. PdxB performs an oxidation reaction using the cofactor NAD. As opposed to most NAD-utilizing enzymes, PdxB is a so-called nicotino-protein that binds NAD tightly (but not covalently). In fact, the enzyme as isolated exists mostly in the reduced NADH-bound state and PdxB can not be oxidized by NAD. This observation raises the question of how the enzyme is reactivated to the oxidized state to perform multiple turnovers. I have discovered that a-ketoglutarate, oxaloacetic acid, and pyruvate, common cellular metabolites, are all capable of re-oxidizing the enzyme-bound NADH. Thus, in a cellular setting PdxB can serve as a useful multi-turnover enzyme.
In PCP degradation my work has focused on the first two enzymes in the degradation pathway, PcpB and PcpD. PcpB converts the PCP substrate to the tetrachlorobenzoquinone (TCBQ), a highly reactive compound that can react non-enzymatically with any number of nucleophiles. PcpD uses TCBQ as a substrate to form the more stable tetrachlorohydroquinone (TCHQ). Both PcpB and PcpD are rather slow and non-specific enzymes, a characteristic often associated with newly evolved enzymes such as these. Thus it was a surprise to find that transfer of the TCBQ intermediate does not occur via solvent, but instead TCBQ is channeled from PcpB to PcpD. Channeling is usually associated with highly specific protein-protein interactions more typical of a well-evolved system. On the other hand, our observation of channeling explains how the highly reactive TCBQ intermediate is restricted from creating havoc inside the cell during PCP degradation.
Rudolph, J., Kim, J., Copley, S.D. (2010) Multiple turnovers of the nicotino-enzyme PdxB require a-keto acids as co-substrates.
Hlouchova, K, Rudolph, J., Pietari, J.M.H., Behlen, L.S. Copley, S.D. (submitted for publication) Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum.