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Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 3(10) helix.

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...

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses.Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel ␤-helical structure yet solved.

Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.