The evolution of enzymes was a critical element of the emergence of life on Earth and its subsequent divergence to occupy a vast range of ecological niches. Most enzymes have impressive catalytic capabilities that have been honed over 3.8 billion years of evolution. We are interested in the early stages of evolution of new enzymes. This application addresses enzymes that appear to have been recently recruited to serve new functions in the pathway for degradation of pentachlorophenol (PCP) in Sphingobium chlorophenolicum, a bacterium isolated from soil heavily contaminated with PCP. S. chlorophenolicum appears to have patched together a poorly functioning pathway for degradation of PCP using enzymes from at least two previously existing pathways. This experimental system provides us with a window on the evolution of a new metabolic pathway at a very early stage in the process. This application focuses on the initial three enzymes in the pathway that limit the flux through the PCP degradation pathway. PCP hydroxylase catalyzes the hydroxylation of PCP to form the toxic intermediate, tetrachlorobenzoquinone (TCBQ). TCBQ reductase catalyzes the reduction of TCBQ to tetrachlorohydroquinone (TCHQ). TCHQ dehalogenase catalyzes two successive reductive dehalogenation reactions that remove chlorines, allowing the ring to be cleaved.
Our aims are 1) to obtain structures of PCP hydroxylase and TCHQ dehalogenase;2) to explore the evolutionary origin and a possible additional function of TCBQ reductase;3) to evolve improved versions of all three enzymes;and 4) to determine how the improved enzymes affect the ability of the bacterium to degrade PCP. The outcome of this work will be a better understanding of the sources from which new enzymes can arise, and the quality of catalysis achieved during the initial stages of evolution of a new enzyme.

Public Health Relevance

We will better understand the prospects for evolution of pathways to degrade toxic pollutants and the problems associated with development of antibiotic resistance by recruitment of enzymes that detoxify antibiotics. In addition, this work will inform efforts to engineer enzymes and bacteria to produce chemicals and pharmaceuticals or to degrade pollutants under environmentally benign conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM078554-03S1
Application #
8055628
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
2010-05-15
Project End
2011-04-30
Budget Start
2010-05-15
Budget End
2011-04-30
Support Year
3
Fiscal Year
2010
Total Cost
$95,718
Indirect Cost
Name
University of Colorado at Boulder
Department
Public Health & Prev Medicine
Type
Other Domestic Higher Education
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Rudolph, Johannes; Erbse, Annette H; Behlen, Linda S et al. (2014) A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum. Biochemistry 53:6539-49
Rokicki, Joe; Knox, David; Dowell, Robin D et al. (2014) CodaChrome: a tool for the visualization of proteome conservation across all fully sequenced bacterial genomes. BMC Genomics 15:65
Yadid, Itamar; Rudolph, Johannes; Hlouchova, Klara et al. (2013) Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol. Proc Natl Acad Sci U S A 110:E2182-90
Copley, Shelley D (2012) Moonlighting is mainstream: paradigm adjustment required. Bioessays 34:578-88
Hlouchova, Klara; Rudolph, Johannes; Pietari, Jaana M H et al. (2012) Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Biochemistry 51:3848-60
Copley, Shelley D (2012) Toward a systems biology perspective on enzyme evolution. J Biol Chem 287:3-10
Copley, Shelley D; Rokicki, Joseph; Turner, Pernilla et al. (2012) The whole genome sequence of Sphingobium chlorophenolicum L-1: insights into the evolution of the pentachlorophenol degradation pathway. Genome Biol Evol 4:184-98
Copley, Shelley D (2009) Evolution of efficient pathways for degradation of anthropogenic chemicals. Nat Chem Biol 5:559-66