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Item Open Access Cloning, expression, purification and characterization of a novel cytochrome P450 isolated from Thermus thermophilus HB27(2002) Blasco, Francesca; Schmid, Rolf D. (Prof. Dr.)Enzymes produced by extremophiles are of considerable biotechnological interest. They show activity and stability at extreme temperatures, low water activity and high hydrostatic pressure. The growing knowledge about "thermozymes" from thermophilic organisms obtained during the recent years and the importance of P450 monooxygenases as potential biocatalysts for industrial applications has created high interest in P450 enzymes from thermophilic bacteria and archaea. Although the homology between P450s is generally low, a few domains and sequence signatures are conserved and can be identified by homology searches. Following this approach a homology search of recently sequenced genomes from thermophiles was performed to identify novel P450 enzymes of this origin. Indeed, a region showing homology to a known bacterial P450 (P450 BM3 from Bacillus megaterium) was found within the Thermus thermophilus HB27 genome, currently being sequenced in Göttingen. Thermus thermophilus is a non-sporulating Gram-negative bacterium isolated from hot springs. Its genome of 1.82 Mbp (excluding the mega-plasmid pTT27 of 0.24 Mbp) has a GC-content of approximately 69 %. The optimal temperature for growth is between 65 and 72 °C, the maximum being 85 °C and the minimum being 47 °C. Bulk protein extracted from this thermophile is much more stable to heat than mesophilic protein extracts; and only about 10 % of the total protein is denatured by heating up to 110 °C for 5 min. Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon monoxide bound form, are a large superfamily of enzymes. P450s are ubiquitous enzymes essential for steroid biosynthesis, catabolism of drugs, utilization of carbon compounds as an energy source in bacteria, and in the production of various macrolide antibiotics. Sequence identity among P450s is often low (normally less than 20 %), but the structural fold has been conserved during evolution. P450s are heme thiolate proteins; the most conserved structural feature is a cysteine as fifth ligand to the heme iron. Normally P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack (often hydroxylation) of non-activated hydrocarbons at physiological temperatures. The substrates of P450s are extremely diverse. They are involved in the biosynthesis of hormones or antibiotics, as well as carcinogenesis and degradation of xenobiotics. The selective oxidation of an unactivated C-H bond in a complex organic molecule to the alcohol functionality (C-OH) has wide applications in chemical synthesis. The present work reports cloning, expression, purification, crystallization and characterization of CYP175A1, a cytochrome P450 from the thermophilic bacterium Thermus thermophilus HB27. For over expression the gene was amplified by PCR in the presence of specific primers from a DNA fragment kindly provided by Prof. H. J. Fritz (University of Göttingen). The PCR product was ligated into the polylinker region of pKK223-3 for expression under control of the tac promoter. The resulting clone was transformed in E. coli BL21(DE3)RP CodonPlus and selected for ampicillin and chloroamphenicol resistance. Expression was induced by IPTG and a yield of approx. 40 mg protein/liter culture was obtained. After cell disruption and ammonium sulfate precipitation P450_Tth was purified to homogeneity, following two different purification protocols. The fractions corresponding to the highest purity were used for crystallization, while the others were used for further biochemical characterization. The 44 kDa protein is soluble and displays an absorption spectrum in the reduced, oxidized, and carbonyl adduct analogous to those of other P450 enzymes. This enzyme exhibits thermostability with a melting temperature 30 °C higher than that of P450cam from P. putida. The crystal structure of CYP175A1 has been determined in co-operation with T. Poulos (University of California, Irvine). CYP175A1 exhibits the typical prism like P450-fold composed of 17 a-helices and 11 b-strands, corresponding to four beta sheets. The location of CYP175A1 in the T. thermophilus genome (close to crtB encoding a phytoene synthase), and the fact that genes for carotenoid biosynthesis occur as a cluster in T. thermophilus as well as in other carotenoid-producing strains, suggested that the gene may be involved in carotenoid biosynthesis. Heterologous complementation using Escherichia coli strains genetically enginereed to produce b-carotene indicated that CYP175A1 was able to catalyze the conversion of b-carotene to zeaxanthin via cryptoxanthin. The model of the b-carotene-CYP175A1 complex was generated. The hydrophobic nature of the ligand fitted well into the binding site of CYP175A1 which contained mostly hydrophobic or neutral residues. The position of the ligand in the active site of the enzyme showed that the C-3 of the b-ionone ring is the closest atom to the heme iron confirming the experimentally observed regioselectivity.