APPLIED MICROBIAL AND CELL PHYSIOLOGY CYP201A2, a cytochrome P450 from Rhodopseudomonas palustris, plays a key role in the biodegradation of tributyl phosphate Cécile Berne & David Pignol & Jérôme Lavergne & Daniel Garcia Received: 9 May 2007 / Revised: 26 July 2007 / Accepted: 31 July 2007 / Published online: 5 September 2007 # Springer-Verlag 2007 Abstract Tributyl phosphate (TBP) is a toxic organophos- phorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosyn- thetic conditions. This study shows that this O 2 - and NADPH/FMNH 2 -dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K d ≈ 65 μM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP. Keywords Bioremediation . Cytochrome P450 . Organophosphorus Introduction Organic phosphorus compounds are essential for the maintenance of life. Nevertheless, the intensive use of organophosphates as pesticides, industrial additives, and chemical warfare agents is responsible for considerable environmental pollution and human health problems, as they act as neurotoxins through a direct or indirect inhibition of acetylcholinesterase (Raushel 2002). The US Environmental Protection Agency approves destruction of these compounds by incineration, but this process is costly, energy-consuming and may result in potentially toxic emissions. As a consequence, many studies have focused on the bioremediation capabilities of microorganisms towards various organophosphates, seeking for rapid, safe, and economical disposal processes for these xenobiotics (Chapalamadugu and Chaudhry 1992; Kumar et al. 1996; Raushel 2002; Singh and Walker 2006). Tributyl phosphate (TBP) is an organophosphate widely used in the composition of defoamers, plasticizers, pesti- cides, hydraulic fluids and as a solvent for conventional nuclear fuel reprocessing, thus, generating considerable wastes. TBP is very stable in natural environments and is poorly degraded by natural photolysis and hydrolysis (Nakamura 1991; US EPA 1992; Hernandez 2002). The degradation of TBP has been little investigated because of its low toxicity to mammals. Nevertheless, in spite of its low solubility in water, TBP is highly toxic to freshwater organisms such as daphnia, gammarus, fish and algae, even at low concentrations (2 to 10 mg l -1 ) (Nakamura 1991; US EPA 1992; Hernandez 2002; Michel et al. 2004). Although Appl Microbiol Biotechnol (2007) 77:135–144 DOI 10.1007/s00253-007-1140-4 C. Berne : D. Pignol : J. Lavergne : D. Garcia (*) DSV/IBEB/SBVME/LBC, UMR 6191 CNRS/CEA/Univ Aix-Marseille, CEA Cadarache, Saint Paul lez Durance F-13108, France e-mail: dgarcia@cea.fr Present address: C. Berne Department of Biology, Indiana University, Bloomington, IN 47405, USA