Cytochrome P450 isolated from Bacillus subtilis (P450 BS␤ ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the ␣-and ␤-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450 BS␤ in the substrate-bound form, determined at a resolution of 2.1 Å. P450 BS␤ exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg 242 guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the ␣-and ␤-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg 242 in the same fashion as has been reported for the active site of chloroperoxidase, His 105 -Glu 183 , which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450 BS␤ is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.Two bacterial cytochrome P450s isolated from Sphingomonas paucimobilis and Bacillus subtilis, P450 SP␣ 1 and P450 BS␤ , respectively, are heme-containing enzymes that catalyze the hydroxylation reaction of long chain fatty acids (e.g. myristic acid) using hydrogen peroxide (H 2 O 2 ) as an oxidant to produce hydroxylated (-OH) fatty acids (1, 2). In the enzymatic reactions, an oxygen atom derived from H 2 O 2 is efficiently introduced into the substrate with a high catalytic turnover (1,000 min Ϫ1 ) (2-4). P450 SP␣ produces the ␣-OH fatty acid (100%) as the product, whereas P450 BS␤ produces both the ␤-OH (60%) and the ␣-OH (40%) fatty acids (1, 2, 4, 5). The amino acid sequence of the two enzymes shares a 44% identity (2). Data base investigation has shown that P450 SP␣ and P450 BS␤ belong to the P450 superfamily and, therefore, they have been given the systematic nomenclature designations CYP152B1 and CYP152A1, respectively (6). However, when compared with reactions catalyzed by other P450s, two characteristic properties in the P450 SP␣ and P450 BS␤ reactions were found, i.e. the utilization of H 2 O 2 and the site specificity of the reaction.In typical P450 reactions an oxygen atom derived from molecular oxygen (O 2 ) is inserted into the substrates (7), and the reaction is referred to as a monooxygenation reaction. Two protons and two electrons are required in the monooxygenation reaction. The electrons are supplied from NAD(P)H through mediation by flavoproteins and iron-sulfur proteins, and the protons are probably delivered from solvent water to the active site through a specific hydrogen-bonding network (8). In the monooxygenase P450 system, H 2 O 2 is sometimes used as a surrogate for the O 2 /2e Ϫ /2H ϩ system (peroxide sh...

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Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-β production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-β mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-β mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.

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