A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803.

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.

[1]  H. Beinert,et al.  Spin-state changes in cytochrome P-450cam on binding of specific substrates. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S Omura,et al.  Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[3]  F Peter Guengerich,et al.  Cytochrome P450: What Have We Learned and What Are the Future Issues? , 2004, Drug metabolism reviews.

[4]  J. Andersen,et al.  Substrate specificity of 6-deoxyerythronolide B hydroxylase, a bacterial cytochrome P450 of erythromycin A biosynthesis. , 1993, Biochemistry.

[5]  C. Khosla,et al.  Cloning and heterologous expression of the epothilone gene cluster. , 2000, Science.

[6]  M. Berenbaum,et al.  Structural and functional divergence of insect CYP6B proteins: From specialist to generalist cytochrome P450. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Y. Fujii‐Kuriyama,et al.  Electron spin resonance studies of wild-type and mutant cytochromes P-450d: effects of mutations at proximal, aromatic and distal sites on g values. , 1990, Biochimica et biophysica acta.

[8]  M. J. Coon,et al.  EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding. , 2000, Biochemical and Biophysical Research Communications - BBRC.

[9]  J. Spudich,et al.  Anabaena Sensory Rhodopsin: A Photochromic Color Sensor at 2.0 Å , 2004, Science.

[10]  J D Lipscomb,et al.  Electron paramagnetic resonance detectable states of cytochrome P-450cam. , 1980, Biochemistry.

[11]  C. Jefcoate,et al.  Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. , 1978, Methods in enzymology.

[12]  M. Schuler,et al.  Functional genomics of P450s. , 2003, Annual review of plant biology.

[13]  S. Sligar,et al.  Oxygenase-catalyzed biological hydroxylations. , 1975, Annual review of biochemistry.

[14]  T. Perlmann,et al.  Retinoid metabolism: a balancing act , 2002, Nature Genetics.

[15]  R. Feyereisen.  Insect P450 enzymes. , 1999, Annual review of entomology.

[16]  Søren Bak,et al.  Comparative Genomics of Rice and Arabidopsis. Analysis of 727 Cytochrome P450 Genes and Pseudogenes from a Monocot and a Dicot1[w] , 2004, Plant Physiology.

[17]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[18]  J. Baudry,et al.  Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling. , 2003, Protein engineering.

[19]  S. Tabata,et al.  Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803. , 1997, Plant & cell physiology.

[20]  O. Gotoh,et al.  Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. , 1992, The Journal of biological chemistry.

[21]  C. Venkatachalam,et al.  LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. , 2003, Journal of molecular graphics & modelling.

[22]  T. Poulos,et al.  Structure of a cytochrome P450-redox partner electron-transfer complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Cupp-Vickery,et al.  Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Berenbaum,et al.  Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase. , 2003, Protein Engineering.

[25]  S. Krasnoff,et al.  Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies , 2002, Journal of bacteriology.

[26]  M. Berenbaum.  Postgenomic Chemical Ecology: From Genetic Code to Ecological Interactions , 2002, Journal of Chemical Ecology.

[27]  F. Guengerich,et al.  Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. , 2001, Chemical research in toxicology.

[28]  S. Sligar.  Nature's universal oxygenases: the cytochromes P450. , 1999, Essays in biochemistry.

[29]  T. Perlmann.  Retinoid metabolism: a balancing act , 2002, Nature Genetics.

[30]  T. Pieler,et al.  Regionalized metabolic activity establishes boundaries of retinoic acid signalling , 1998, The EMBO journal.

[31]  H. Ochi,et al.  Electron paramagnetic resonance studies of the purified cytochrome P-450scc and P-45011beta from bovine adrenocortical mitochondria. , 1979, Biochimica et biophysica acta.