Chlorinated natural products include vancomycin and cryptophycin A. Their biosyntheses involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and FAD are separated by a 10Å-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. Based on biochemical studies, crystal structures and by analogy with monooxygenases, we predict FADH 2 reacts with O 2 making peroxy-flavin which is decomposed by Cl −. The resulting HOCl is guided through the tunnel, to tryptophan, where it is activated to participate in electrophilic aromatic substitution. In addition to man-made chemicals, there are nearly four thousand chlorinated and brominated natural products (1), including drugs such as vancomycin (2), rebeccamycin (3) and cryptophycin A (4). The de-novo chemical synthesis of complex natural products is often too expensive or too difficult to be practical. Their production relies on fermentation and introducing diversity in such molecules requires protein engineering. This has been hampered by a lack of understanding of the molecular basis of the biological regioselective halogenation mechanism. Metal-dependent haloperoxidases were once thought to catalyze all halogenation reactions in biology and fall into two classes. Both, heme-iron-dependent enzymes (5) and vanadium-dependent enzymes (6), have been structurally characterized. Although different in structure, both form a metal-bound hydrogen peroxide, which reacts with halide ions producing a metal-bound hypohalite ion. This ion dissociates from the metal as hypohalous acid (5,6) where, in solution, it reacts with substrate. Such halogenation lacks regioselectivity and substrate specificity (7). Perhydrolases are now recognized not to be involved in halometabolite biosynthesis (8). A new halogenase was reported by Dairi et al. identifying the gene for the chlorinating enzyme in chlorotetracycline biosynthesis (9). The gene product showed no similarity to haloperoxidases. Studies of the antifungal compound pyrrolnitrin from Pseudomonas fluorescens identified two related genes (prnA and prnC), coding for two halogenating enzymes (10) (fig. S1). Both contain a flavin binding site (9-13) and exhibit weak sequence homology to flavin-dependent monooxygenase enzymes (14). PrnA catalyzes the regioselective chlorination of the 7-position of tryptophan (15). Turnover requires that FAD is reduced to FADH 2 (by flavin reductase) and that O 2 is present (11). One sentence: The crystal structure of tryptophan 7-halogenase indicates that the halogenation of important metabolites in bacteria proceeds by the novel use of hypohalous acid to achieve regioselective substitution.

3Outer membrane proteins (OMPs) play important roles in Gram-negative bacteria, mitochondria and chloroplasts in nutrition transport, protein import, secretion, and other fundamental biological processes [1][2][3] . Dysfunction of mitochondria outer membrane proteins are linked to disorders such as diabetes, Parkinsons and other neurodegenerative diseases 4,5 . The OMPs are inserted and folded correctly into the outer membrane (OM) by the conserved OMP85 family proteins [6][7][8] , suggesting that similar insertion mechanisms may be used in Gram-negative bacteria, mitochondria and chloroplasts.In Gram-negative bacteria, OMPs are synthesized in the cytoplasm, and are transported across the inner membrane by SecYEG into the periplasm 8,9 . The seventeen kilodalton (kDa) protein (Skp) and the survival factor A (SurA) chaperones escort the unfolded OMPs across the periplasm to the β-barrel assembly machinery (BAM), which is responsible for insertion and assembly of OMPs into the OM 10-12 . InEscherichia coli, the BAM complex consists of BamA and four lipoprotein subunits, BamB, BamC, BamD and BamE. BamA is comprised of five N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal OMP transmembrane barrel, while the four lipoproteins are affixed to the membrane by N-terminal lipid-modified cysteines. Of these subunits, BamA and BamD are essential 3,6 . One copy of each of these five proteins is required to form the BAM complex with an approximate molecular weight of 200 kDa (Extended Data Fig. 1). In vitro reconstitution of the E.coli BAM complex and functional assays showed that all five subunits are required to obtain the maximum activity of BAM [13][14][15][16] . Furthermore, comparison of the two complexes reveals that the periplasmic units are rotated with respect to the barrel, which appears to be linked to significant conformational changes in the β-strands β1C-β6C of the barrel. Taken together this suggests a novel insertion mechanism whereby rotation of the BAM periplasmic ring promotes insertion of OMPs into the OM. To our knowledge, this is the first reported crystal structure of an intramembrane barrel with a lateral-open conformation.Unique architecture of two E. coli BAM complexes X-ray diffraction data of selenomethionine labelled crystals were collected to 3.9Ångström (Å) resolution and the BAM structure was determined by singlewavelength anomalous dispersion (SAD) and manual molecular replacement (Methods, Extended Data Table 1). The first structure contained four proteins: BamA, BamC, BamD and BamE (Fig. 1a-c), with the electron density and crystal packing indicating that the BamB is absent in the complex. This was confirmed by SDS-PAGE analysis of the crystals (Extended Data Fig. 1 and Supplementary Data Fig. S1). In this model, BamA, BamC, BamD and BamE contain residues E22-I806, C25-K344, E26-S243, and C20-E110, respectively. The machinery is approximately 115 Å in length, 84 Å in width and 132 Å in height (Fig. 1a). 5The architecture of BamACDE resembles a top hat with a...

Summary Lassa fever virus (LASV) causes thousands of deaths yearly and is a biological threat agent, for which there is no vaccine and limited therapy1. The nucleoprotein (NP) of LASV plays essential roles in viral RNA synthesis and immune suppression2-6, the molecular mechanisms of which are poorly understood. Here, we report the crystal structure of LASV NP at 1.80 Angstrom resolution, which reveals N- and C-domains with structures unlike any of the reported viral NPs7-10. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3′-5′ exoribonuclease activity involved in suppressing interferon induction. This is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and suggests unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.

Pathogenic bacteria frequently cloak themselves with a capsular polysaccharide layer. Escherichia coli group 1 capsules are formed from repeat-unit polysaccharides with molecular weights exceeding 100 kDa. The export of such a large polar molecule across the hydrophobic outer membrane in Gram-negative bacteria presents a formidable challenge, given that the permeability barrier of the membrane must be maintained. We describe the 2.26 Å structure of Wza, an integral outer membrane protein, that is essential for capsule export. Wza is an octamer, with a composite molecular weight of 340 kDa, and it forms an "amphora"-like structure. The protein has a large central cavity 100 Å long and 30 Å wide. The transmembrane region is a novel α-helical barrel, and is linked to three additional novel periplasmic domains, marking Wza as the representative of a new class of membrane protein.Although Wza is open to the extracellular environment, a flexible loop in the periplasmic region occludes the cavity and may regulate the opening of the channel. The structure defines the route taken by the capsular polymer as it exits the cell, using the structural data we propose a mechanism for the translocation of the large polar capsular polysaccharide.