Structure and Function of a Cytochrome P450 From Synechocystis Sp. PCC 6803
Ke, Na
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https://hdl.handle.net/2142/86702
Description
Title
Structure and Function of a Cytochrome P450 From Synechocystis Sp. PCC 6803
Author(s)
Ke, Na
Issue Date
2007
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
"Three decades ago it was thought that there were but a few cytochrome P450s and their functional landscape was relatively limited. We now know that there are over 6,000 P450 gene products, which display a variety of activities and provide interesting and crucial metabolic transformations. The search for novel functions and the revelation of new structures thus demands exploration of cytochrome P450s in novel econiches. Cyanobacteria, photosynthetic bacteria, are thought of as the ancestors of chloroplasts. They produced oxygen in the primitive atmosphere, representing an interesting, evolutionarily ""old"" bacterial group. Over 60 cytochrome P450 or cytochrome P450-like genes have been identified in 22 cyanobacterial genomes. The universal distribution of P450s in cyanobacteria suggests that these P450s carry out important biological and metabolic functions. Although from an evolutionary point of view the cyanobacterial P450s provide an important link between bacteria and plant P450 monoxygenases, to date none of these P450s has been functionally characterized. Addressing this lacuna, the cyp120A1 gene from Synechocystis sp. PCC 6803 has been cloned, heterologously expressed in and purified from E. coli. Homology modeling of CYP120A1 based on the CYP1O7A structure and subsequent in silico docking of a small molecule library identified all-trans retinoic acid as a potential substrate. Optical difference and electron paramagnetic resonance (EPR) spectroscopy show that retinoic acid binds to CYP120A1 with a high affinity. The structures of substrate-free and retinoic acid-bound CYP120M have been determined to a resolution of 2.4 A and 2.1 A, which provides new insight regarding P450 and its substrate interactions. The biological function of CYP120A1 was investigated through RT-PCR analysis of gene expression under different growth conditions as well as by generating and phenotyping a cyp120A1 knockout strain. Thus the research project described herein forms a foundation for understanding the structure and function of cyanobacterial cytochrome P450s."
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