Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases.

Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen Pseudomonas aeruginosa in which they play an essential role in host infection. PVD biosynthesis encompasses a complex pathway comprising cytosolic nonribosomal peptide synthetases that produce a polypeptide precursor that periplasmic enzymes convert to the final product. The structures of most enzymes involved in PVD chromophore maturation have been elucidated, but the structure of the essential tyrosinase PvdP, a monooxygenase required for the penultimate step in PVD biosynthesis, is not known. Here, we closed this gap by determining the crystal structure of PvdP in an apo and tyrosine-complexed state at 2.1 and 2.7 Å, respectively. These structures revealed that PvdP is a homodimer, with each chain consisting of a C-terminal tyrosinase domain and an N-terminal eight-stranded ß-barrel reminiscent of streptavidin that appears to have a structural role only. We observed that ligand binding leads to the displacement of a "placeholder" tyrosine that blocks the active site in the apo structure. This exposes a large, deep binding site that seems suitable for accommodating ferribactin, a substrate of PvdP in PVD biosynthesis. The binding site consists almost exclusively of residues from the tyrosinase domain. Of note, we also found that this domain is more closely related to tyrosinases from arthropods rather than to tyrosinases from other bacteria. In conclusion, our work unravels the structural basis of PvdP's activity in PVD biosynthesis, observations that may inform structure-guided development of PvdP-specific inhibitors to manage P. aeruginosa infections.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

The Structure of LiuC, a 3-Hydroxy-3-Methylglutaconyl CoA Dehydratase Involved in Isovaleryl-CoA Biosynthesis in Myxococcus xanthus, Reveals Insights into Specificity and Catalysis.

Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3-methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Šof LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3-hydroxy-3-methylglutaconyl CoA to 3-methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid-base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl-CoA is a source for isobutene, a precursor for renewable fuels and chemicals.

In Silico Prediction of Target-Inhibitor Interaction.

For a long time people are trying to predict interactions of molecules. The base for this is always a 3D structure of both partners. Based on the increasing numbers of X-Ray structures of proteins with and without bounded molecules algorithms were developed to predict the target-inhibitor interaction based on known structures of proteins. The 3D structure of inhibitors may be based on known structures or even composed during the prediction process.In most cases the problem is to find a molecule that could inhibit the function of a protein. In a second step the structure of the "inhibitor" has to be optimized.There are a lot of limitations using these technics to find a usable inhibitor, as in real world experiments the inhibitor shall only inhibit this protein and not knock out others essential functions of the cell or, in case of medical applications the host organism.

Permissivity of the biphenyl-specific aerobic bacterial metabolic pathway towards analogues with various steric requirements.

It has repeatedly been shown that aryl-hydroxylating dioxygenases do not possess a very high substrate specificity. To gain more insight into this phenomenon, we examined two powerful biphenyl dioxygenases, the well-known wild-type enzyme from Burkholderia xenovorans LB400 (BphA-LB400) and a hybrid enzyme, based on a dioxygenase from Pseudomonas sp. B4-Magdeburg (BphA-B4h), for their abilities to dioxygenate a selection of eight biphenyl analogues in which the second aromatic ring was replaced by aliphatic as well as aliphatic/aromatic moieties, reflecting a variety of steric requirements. Interestingly, both enzymes were able to catalyse transformation of almost all of these compounds. While the products formed were identical, major differences were observed in transformation rates. In most cases, BphA-B4h proved to be a significantly more powerful catalyst than BphA-LB400. NMR characterization of the reaction products showed that the metabolite obtained from biphenylene underwent angular dioxygenation, whereas all other compounds were subject to lateral dioxygenation at ortho and meta carbons. Subsequent growth studies revealed that both dioxygenase source strains were able to utilize several of the biphenyl analogues as sole sources of carbon and energy. Therefore, prototype BphBCD enzymes of the biphenyl degradative pathway were examined for their ability to further catabolize the lateral dioxygenation products. All of the ortho- and meta-hydroxylated compounds were converted to acids, showing that this pathway is quite permissive, enabling catalysis of the turnover of a fairly wide variety of metabolites.

Simian immunodeficiency virus and human immunodeficiency virus type 1 matrix proteins specify different capabilities to modulate B cell growth.

UNLABELLED: Exogenous HIV-1 matrix protein p17 (p17) deregulates the function of different cells after its N-terminal loop (AT20) binding to the chemokine receptors CXCR1 and CXCR2. One site within AT20 has been recently found to be the major determinant of viral fitness following transmission of simian immunodeficiency virus (SIV) to the human host. Therefore, we sought to determine whether SIV matrix protein (MA) was already capable of interacting with CXCR1 and CXCR2 and mimic p17 biological activities rather than this being a newly acquired function during host adaptation. We show here that SIV MA binds with the same affinity of p17 to CXCR1 and CXCR2 and displays both p17 proangiogenic on human primary endothelial cells and chemotactic activity on human primary monocytes and B cells. However, SIV MA exhibited a higher degree of plasticity than p17 in the C terminus, a region known to play a role in modulating B cell growth. Indeed, in contrast to p17, SIV MA was found to activate the phosphatidylinositol 3-kinase/Akt signaling pathway and strongly promote B cell proliferation and clonogenic activity. Interestingly, we have recently highlighted the existence of a Ugandan HIV-1 strain-derived p17 variant (S75X) with the same B cell growth-promoting activity of SIV MA. Computational modeling allowed us to hypothesize an altered C terminus/core region interaction behind SIV MA and S75X activity. Our findings suggest the appearance of a structural constraint in the p17 C terminus that controls B cell growth, which may help to elucidate the evolutionary trajectory of HIV-1. IMPORTANCE: The HIV-1 matrix protein p17 (p17) deregulates the biological activities of different cells after binding to the chemokine receptors CXCR1 and CXCR2. The p17 functional domain responsible for receptors interaction includes an amino acid which is considered the major determinant of SIV replication in humans. Therefore, we sought to determine whether SIV matrix protein (SIV MA) already had the ability to bind to both chemokine receptors rather than being a function newly acquired during host adaptation. We show here that SIV MA binds to CXCR1 and CXCR2 and fully mimics the p17 proangiogenic and chemokine activity. However, it differs from p17 in its ability to signal into B cells and promote B cell growth and clonogenicity. Computational analysis suggests that the accumulation of mutations in the C-terminal region may have led to a further SIV MA adaptation to the human host. This finding in turn sheds light on the evolutionary trajectory of HIV-1.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.

Structure and function of enzymes in heme biosynthesis.

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the "Radical SAM enzyme" coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.

Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder.

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-A resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.

BRAGI: linking and visualization of database information in a 3D viewer and modeling tool.

BRAGI is a well-established package for viewing and modeling of three-dimensional (3D) structures of biological macromolecules. A new version of BRAGI has been developed that is supported on Windows, Linux and SGI. The user interface has been rewritten to give the standard 'look and feel' of the chosen operating system and to provide a more intuitive, easier usage. A large number of new features have been added. Information from public databases such as SWISS-PROT, InterPro, DALI and OMIM can be displayed in the 3D viewer. Structures can be searched for homologous sequences using the NCBI BLAST server.

Mechanistic and molecular investigations on stabilization of horseradish peroxidase C.

The enzyme horseradish peroxidase (HRP) shows a decreasing activity when the enzyme's substrate hydrogen peroxide is present with the degree of inactivation being dependent on the incubation time and the hydrogen peroxide concentration. Incubation times of some minutes do not inactivate the enzyme independent of the H2O2 concentration. After several hours, only 50% of the activity is found for a medium H2O2 excess, and a >100-fold excess of H2O2 completely inactivates the enzyme. Polymeric additives, in particular Gafquat, lead to higher residual activities, whereas stabilizers, such as aminopyrine, preserve the full activity. Circular dichroism (CD) measurements reveal that the enzyme structure remains more or less unchanged when hydrogen peroxide is added. Only when a 1000-fold excess of hydrogen peroxide is present are structural changes observed. UV spectra highlight that the heme group in the enzyme is affected by hydrogen peroxide in a first step. Without any prolonged incubation, a decrease of the Soret band to approximately 50% is found for low hydrogen peroxide concentrations (HRP/H2O2 from 1:1 to 1:100). Higher H2O2 concentrations lead to the formation of catalytically inactive HRP forms. Preincubation of Gafquat, which is a copolymer from vinylpyrrolidone and derivatized methyl methacrylate, with hydrogen peroxide shifts the influence of hydrogen peroxide to higher concentrations, the shift being dependent on the Gafquat concentration. This effect is not observed for other polymers, such as dextrans, but it is also found for the stabilizer aminopyrine. Extended incubation times (24 h) of HRP together with H2O2, however, lead to an at least partial recovery of the Soret band for lower H2O2 concentrations (H2O2/HRP from 1:1 to 1:100). When hydrogen peroxide is used in a >100 fold excess, the heme group is irreversibly destroyed, and even the characteristic band of cpd III is not found. Here, the presence of Gafquat only reduces the degree of destruction. Computer modeling of the interaction between the polymers and the enzyme shows no specific binding sites for the functional groups of the vinylpyrrolidone-methacrylate copolymer Gafquat or of DEAE-dextran on the enzyme, whereas for the only activating polymer, polyethylenimine clustering of binding sites is observed.