Crystal structures reveal how distinct sites on the cysteine desulfurase IscS bind two different sulfur-acceptor proteins, IscU and TusA, to transfer sulfur atoms for iron-sulfur cluster biosynthesis and tRNA thiolation.
The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central alpha-helix extending 100 A into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery.
Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.
HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of L-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn 2؉ ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg 2؉ , Mn 2؉ , Co 2؉ , or Zn 2؉ , but Ca 2؉ has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg 2؉ ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca 2؉ .The histidine biosynthetic pathway serves as a model system for better understanding of the fundamental metabolic, physiological, and genetic processes in bacteria (1). This pathway is identical in both Escherichia coli and Salmonella typhimurium and has been thoroughly characterized (1, 2). The sixth and eighth steps of histidine biosynthesis are catalyzed by imidazole glycerol phosphate dehydratase (IGPD, 3 EC 4.2.1.19) and histidinol phosphate phosphatase (HPase, EC.3.1.3.15) respectively (1) (Scheme 1). In protobacteria, including E. coli and S. typhimurium, the IGPD and HPase activities are encoded by a single gene (3-5), whereas in archaea, eukarya, and most bacteria they are encoded by two separate genes (3). The bifunctional HisB enzyme has been proposed to be the result of a fusion of two independent cistrons that occurred recently in evolution (3).Biochemical and genetic studies of the HisB enzyme together suggest that both of its enzymatic activities are independent of one another and reside in separate domains (6 -8). The HPase activity is found within the N-terminal domain (residues 1-167, HisB-N), whereas the C-terminal domain (residues 168 -356) exhibits the IGPD activity. The phosphatase activity requires the presence of metal ions such as Mg 2ϩ , Mn 2ϩ , Co 2ϩ , or Zn 2ϩ but is inhibited by calcium (9). Based on the presence of four invariant aspartic acid residues, HisB has been classified as a member of the haloacid dehalogenase-like hydrolase (HAD) family within the "DDDD" superfamily of aspartyl-phosphate utilizing phosphohydrolases/phosphotransferases (10, 11). The phosphoryl transfer catalyzed by phosphotransferases and phosphatases from the HAD family occurs via a phosphoa...
Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido-2,4,6-trideoxy-d-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 A resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal alpha/beta-domain and a C-terminal left-handed beta-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the beta-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the beta-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.
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