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Ergothioneine is a sulfur-containing histidine derivative synthesized by many bacteria and most fungi but it also finds its way into human tissue by way of specific absorption from the diet. The precise role of ergothioneine is not yet known but there is growing evidence that it plays a role as an antioxidant protecting human cells from oxidative stress and pathogenic bacteria from host defenses. In this review we highlight recent advances in understanding the structural basis of ergothioneine biosynthesis. In addition to unusual carbon–sulfur bond forming enzymology this research has revealed that ergothioneine biosynthesis has emerged at least three times by independent molecular evolution. [ABSTRACT FROM AUTHOR]

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd1 nitrite reductase NirS in, for example, the Gram‐negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight‐bladed β‐propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d1‐bound form. The heme d1‐free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d1‐binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed. [ABSTRACT FROM AUTHOR]

Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines. [ABSTRACT FROM AUTHOR]

The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis. [ABSTRACT FROM AUTHOR]

The stable post-translational modification of proteins by adenylylation or uridylylation was discovered more than four decades ago as a mechanism to regulate the activity of enzymes. Although many other processes involving the covalent transfer of an AMP residue to an amino acid side chain have been identified since then, these are transient adenylylation events that essentially use the free energy of ATP hydrolysis to activate specific processes. Recently, new examples of stable adenylylation of small GTPases involved in signal transduction and regulation of cellular events were discovered, which appear to modulate downstream processes such as cytoskeletal rearrangement and vesicular trafficking. We present a survey of the historical and modern phases of research in this area, focusing on the common and differing aspects of protein adenylylation. [Copyright &y& Elsevier]

Myosin II is the major component of the muscle thick filament, It consists of two N-terminal S1 subfragments (‘heads’) connected to a long dimeric coiled-coil rod. The rod is in itself twofold symmetric, but in the filament, the two heads point away from the filament surface and are therefore not equivalent. This breaking of symmetry requires the initial section of the rod, subfragment 2 (S2), to be relatively flexible. S2 is an important functional element, involved in various mechanisms by which the activity of smooth and striated muscle is regulated. We have determined crystal structures of the 126 N-terminal residues of S2 from human cardiac β-myosin II (S2-Δ), of both WT and the disease-associated E924K mutant. S2-Δ is a straight parallel dimeric coiled coil, but the N terminus of one chain is disordered in WT-S2-Δ due to crystal contacts, indicative of unstable local structure. Bulky noncanonical side chains pack into a/d positions of S2-Δ's N terminus, leading to defined local asymmetry and axial stagger, which could induce nonequivalence of the S1 subfragments. Additionally, S2 possesses a conserved charge distribution with three prominent rings of negative potential within S2-Δ the first of which may provide a binding interface for the ‘blocked head’ of smooth muscle myosin in the OFF state. The observation that many disease-associated mutations affect the second negatively charged ring further suggests that charge interactions play an important role in regulation of cardiac muscle activity through myosin-binding protein C. [ABSTRACT FROM AUTHOR]

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 Å. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed. [ABSTRACT FROM AUTHOR]

MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 m M KF, 14%( w/ v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a =  b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%( v/ v) glycerol and data have been collected to 2.7 Å resolution. [ABSTRACT FROM AUTHOR]

Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, anti-tumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-d i hydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function. [ABSTRACT FROM AUTHOR]

dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyzes the final step in the conversion of dTDP-D-glucose to dTDP-L-rhamnose in an NAD(P)H- and Mg2+-dependent reaction. L-rhamnose biosynthesis is an antibacterial target. The structure of RmlD from Salmonella enterica serovar Typhimurium has been determined, and complexes with NADH, NADPH, and dTDP-L-rhamnose are reported. RmlD differs from other short chain dehydrogenases in that it has a novel dimer interface that contains Mg2+. Enzyme catalysis involves hydride transfer from the nicotinamide ring of the cofactor to the C4′-carbonyl group of the substrate. The substrate is activated through protonation by a conserved tyrosine. NAD(P)H is bound in a solvent-exposed cleft, allowing facile replacement. We suggest a novel role for the conserved serine/threonine residue of the catalytic triad of SDR enzymes. [Copyright &y& Elsevier]

The synthesis of deoxy-thymidine di-phosphate (dTDP)-L-rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP-L-rhamnose biosynthetic pathway is glucose-1-phosphate thymidylyltransferase (Rm1A). Rm1A is inhibited by dTDP—L-rhamnose thereby regulating L-rhamnose production in bacteria. The structure of Pseudomonas aeruginosa Rm1A has been solved to 1.66 Å resolution. Rm1A is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to Rm1A-like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate-product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP—L-rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Reports on the elucidation of the structure in water and additionally in 50 percent trifluoroethanol (TFE) solution of helodermin, by 2D H nuclear magnetic resistance (NMR) spectroscopy. Calculation of detailed structures from the qualitative NOE data; Definition of regions of stable secondary structure from peptide conformations.

Until quite recently, high-level expression of full-length cellular prion protein (Prpc) in bacterial cells was not possible. We describe here the effective purification of mature Syrian golden hamster PrPc (residues 23-231) as a C-terminal fusion to glutathione S-transferase (GST) from inclusion bodies expressed in Escherichia coli. Purification of the denatured fusion protein was simplified greatly by the introduction of a C-terminal histidine anchor, leading to 255 mg pure GST-PrPc-His6/l bacterial broth, which could be refolded easily by dilution in 20 mM Tris, 5 mM dithiothreitol, 1 mM EDTA, pH 9.0. Refolding was monitored by following GST activity. Mature Syrian hamster PrPc (residues 23-231) was released from the refolded fusion protein by thrombin digestion, yielding 73 mg homogeneous protein/l bacterial culture after purification. The recombinant protein was identified by monoclonal antibodies, Edman sequencing and matrix-assisted laser-desorption/ionization MS. Correct folding was confirmed by near-ultraviolet circular dichroism spectroscopy. Samples resulting from different purification steps were sensitive to proteinase K digestion and showed no signs of infectivity in animal experiments, demonstrating that the PrPc produced is identical with the cellular isoform. The presented purification procedure should prove useful for the production of other GST-fusion proteins. [ABSTRACT FROM AUTHOR]

A major drawback for practical application of halohydrin dehalogenase HheG in biocatalysis is its rather low thermal stability and low organic solvent tolerance. We therefore pursued a stabilization of HheG via immobilization as cross‐linked enzyme crystals. Since glutaraldehyde inactivates HheG, we introduced a cysteine residue in the crystal interface, which enabled thiol‐specific cross‐linking at well‐defined cross‐linking sites. Variant HheG D114C displayed improved crystallizability and yielded stable and catalytically active CLECs using bis‐maleimidoethane as cross‐linker. Effective cross‐linking at the predefined site could be confirmed via the CLEC crystal structure. Compared to soluble enzyme, the CLECs displayed significantly improved stability and activity at higher temperatures, lower pH values and in the presence of water‐miscible organic solvents, which enabled their reuse over 21 days in the azidolysis of cyclohexene oxide. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, the causative agent of a life‐threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella‐containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB‐secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/β‐hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate‐binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA. [ABSTRACT FROM AUTHOR]

Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2'-OH of RNA. The central 'guide RNA' has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C'/D' motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C'/D' motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2'-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C'/D' motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2'-O-methylation enzymes. [ABSTRACT FROM AUTHOR]

The resolution of 3D electron diffraction (ED) data of small‐molecule crystals is often relatively poor, due to either electron‐beam radiation damage during data collection or poor crystallinity of the material. Direct methods, used as standard for crystal structure determination, are not applicable when the data resolution falls below the commonly accepted limit of 1.2 Å. Therefore an evaluation was carried out of the performance of molecular replacement (MR) procedures, regularly used for protein structure determination, for structure analysis of small‐molecule crystal structures from 3D ED data. In the course of this study, two crystal structures of Bi‐3812, a highly potent inhibitor of the oncogenic transcription factor BCL6, were determined: the structure of α‐Bi‐3812 was determined from single‐crystal X‐ray data, the structure of β‐Bi‐3812 from 3D ED data, using direct methods in both cases. These data were subsequently used for MR with different data types, varying the data resolution limit (1, 1.5 and 2 Å) and by using search models consisting of connected or disconnected fragments of BI‐3812. MR was successful with 3D ED data at 2 Å resolution using a search model that represented 74% of the complete molecule. [ABSTRACT FROM AUTHOR]

The crystal structure of the 268‐residue periplasmic protein PA1624 from the opportunistic pathogen Pseudomonas aeruginosa PAO1 was determined to high resolution using the Se‐SAD method for initial phasing. The protein was found to be monomeric and the structure consists of two domains, domains 1 and 2, comprising residues 24–184 and 185–268, respectively. The fold of these domains could not be predicted even using state‐of‐the‐art prediction methods, and similarity searches revealed only a very distant homology to known structures, namely to Mog1p/PsbP‐like and OmpA‐like proteins for the N‐ and C‐terminal domains, respectively. Since PA1624 is only present in an important human pathogen, its unique structure and periplasmic location render it a potential drug target. Consequently, the results presented here may open new avenues for the discovery and design of antibacterial drugs. [ABSTRACT FROM AUTHOR]

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase‐coupled NADH‐dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three‐component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe–4S]‐cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD‐binding domain, an NAD‐binding domain and a C‐terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD‐ and NAD‐binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD‐binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD‐binding site of FdR9 contains a modified Rossmann‐fold motif, GxSxxS, instead of the classical GxGxxG motif. [ABSTRACT FROM AUTHOR]

Monoheme c‐type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α‐helices that surround a covalently attached heme. An intriguing feature of many monoheme c‐type cytochromes is their capacity to form oligomers by exchanging at least one of their α‐helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c‐type cytochrome co‐encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe‐SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain‐swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c‐type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homosapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in‐depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine‐based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X‐ray crystal structure in complex with PqsR facilitated further structure‐guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof‐of‐principle for adjunctive treatment scenarios. [ABSTRACT FROM AUTHOR]

Isovaleryl coenzyme A (IV-CoA) performs a crucial role during development and fruiting-body formation in myxobacteria, which is reflected in the existence of a de novo biosynthetic pathway that is highly upregulated when leucine, the common precursor of IV-CoA, is limited. The final step in de novo IV-CoA biosynthesis is catalyzed by AibC, a medium-chain dehydrogenase/reductase. Here, the crystal structure of AibC from Myxococcus xanthus refined to 2.55 Å resolution is presented. The protein adopts two different conformations in the crystal lattice, which is a consequence of partial interaction with the purification tag. Based on this structure, it is suggested that AibC most probably uses a Zn2+-supported catalytic mechanism in which NADPH is preferred over NADH. Taken together, this study reveals structural details of the alternative IV-CoA-producing pathway in myxobacteria, which may serve as a base for further biotechnological research and biofuel production. [ABSTRACT FROM AUTHOR]

The crystal structure of the natural product argyrin D was determined from 3D electron diffraction data. The molecular conformation of the newly determined structure is similar to that of other argyrins. We attribute the exceptional stability of this geometry to an extended intramolecular hydrogen-bonding network. [ABSTRACT FROM AUTHOR]

Leishmania species are early branching eukaryotic parasites that cause difficult-to-treat tissue-damaging diseases known as leishmaniases. As a hallmark of their parasitic lifestyle, Leishmaniae express a number of aminotransferases that are involved in important cellular processes and exhibit broader substrate specificity than their mammalian host's counterparts. Here, we have determined the crystal structure of the broad specificity aminotransferase from Leishmania mexicana (LmexBSAT) at 1.91 Å resolution. LmexBSAT is a homodimer and belongs to the α-branch of family-I aminotransferases. Despite the fact that the protein was crystallized in the absence of substrates and has lost the pyridoxal-5′-phosphate (PLP) cofactor during crystallization, the structure resembles the closed, ligand-bound form of related enzymes such as chicken cytosolic aspartate aminotransferase. Its broader substrate specificity seems to be rooted in increased flexibility of a substrate-binding arginine (R291) and the interactions of this residue with the N-terminus of the second chain of the dimer. [ABSTRACT FROM AUTHOR]

The non-heme iron enzyme EgtB catalyzes O2-dependent CS bond formation between γ-glutamyl cysteine and N-α-trimethyl histidine as the central step in ergothioneine biosynthesis. Both, the catalytic activity and the architecture of EgtB are distinct from known sulfur transferases or thiol dioxygenases. The crystal structure of EgtB from Mycobacterium thermoresistibile in complex with γ-glutamyl cysteine and N-α-trimethyl histidine reveals that the two substrates and three histidine residues serve as ligands in an octahedral iron binding site. This active site geometry is consistent with a catalytic mechanism in which CS bond formation is initiated by an iron(III)-complexed thiyl radical attacking the imidazole ring of N-α-trimethyl histidine. [ABSTRACT FROM AUTHOR]

Das eisenhaltige Nicht-Häm-Enzym EgtB katalysiert die sauerstoffabhängige Bildung einer Kohlenstoff-Schwefel-Bindung zwischen γ-Glutamylcystein und N-α-Trimethylhistidin als zentralen Schritt der Ergothionein-Biosynthese. Sowohl die katalytische Aktivität als auch die 3D-Struktur von EgtB unterscheiden sich von bisher bekannten Schwefel-Transferasen oder Thiol-Dioxygenasen. Die Kristallstruktur von EgtB aus Mycobacterium thermoresistibile im Komplex mit γ-Glutamylcystein und N-α-Trimethylhistidin zeigt die zwei Substrate und drei Histidinreste als Liganden der oktaedrischen Eisenbindestelle. Diese Geometrie des aktiven Zentrums passt zu einem katalytischen Mechanismus, in dem die Bildung der Kohlenstoff-Schwefel-Bindung von einem Eisen(III)-komplexierten Thiyl-Radikal initiiert wird, indem dieses den Imidazolring des N-α-Trimethylhistidins angreift. [ABSTRACT FROM AUTHOR]

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α), N(α), N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Abstract: The positive transcription elongation factor P-TEFb mediates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb is negatively regulated by the cellular protein Hexim1 (hexamethylene bisacetamide-inducible protein 1), which is highly conserved in higher eukaryotes. The C-terminal coiled-coil domain of Hexim1 recognizes the Cyclin T subunit of P-TEFb, whereas a central PYNT motif is required to inhibit the cyclin-dependent kinase Cdk9 by a yet unknown mechanism. Here, the crystal structure of the Cyclin T-binding domain (TBD) of human Hexim1 was determined at 2.1 Å resolution using a deletion mutant of three residues in its central stammer motif. The structure showed a continuous parallel coiled-coil domain of nine hepta-repeats with a preceding helix encompassing up to 15 residues. Two uncommon residues at heptad a positions in the N-terminal part of the coiled-coil structure, Lys284 and Tyr291, stabilize the preceding helix by a tight intermolecular hydrogen bond network with residues of the opposing chain. These interactions delineate a characteristic turn between both helices that is supposed to mediate binding to Cyclin T1. Stabilization of the coiled-coil domain by deletion of the stammer region was confirmed by NMR spectroscopic and backbone dynamic analyses analyzing wild-type TBD and three mutant variants. This study thus provides structural insights into the recognition of the regulator protein Hexim1 by P-TEFb and the modulation of coiled-coil dynamics by specific discontinuities. [Copyright &y& Elsevier]

Mutations in the gene encoding smooth muscle cell alpha actin (ACTA2) have recently been shown to cause familial thoracic aortic aneurysms leading to type A dissections (TAAD) and predispose to premature stroke and coronary artery disease. In order to further explore the role of ACTA2 variations in the pathogenesis of TAAD, we sequenced the coding regions of this gene in 40 unrelated German patients with TAAD (with (n=21) or without (n=19) clinical features suggestive of Marfan syndrome). All patients had previously tested negative for mutations in the FBN1 and TGFBR2 genes. We identified three novel ACTA2 mutations and mapped them on a three-dimensional model of actin. Two mutations affect residues within (M49V) or adjacent to (R39C), the DNAse-I-binding loop within subdomain 2 of alpha actin. They were observed in families with recurrent aortic aneurysm (R39C) or aortic dissection (M49V). The third mutation causes an exchange in the vicinity of the ATP-binding site (G304R) in a patient thought to have isolated TAAD. None of the affected individuals had clinical features typical for Marfan syndrome, and no case of premature stroke or coronary artery disease was reported from the affected families. In conclusion, we underscore the role of ACTA2 mutations in nonsyndromic TAAD and suggest that ACTA2 should be included in the genes routinely investigated for syndromic and nonsyndromic TAAD. Detailed clinical investigations of additional families are warranted to further explore the full range of phenotypic signs associated with the three novel mutations described here. [ABSTRACT FROM AUTHOR]

Summary: Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity. [Copyright &y& Elsevier]

Die nicht‐kanonische Terpencyclase AsR6 ist für die Bildung von 2E,6E,9E‐Humulen während der Biosynthese des Tropolonsesquiterpenoids (TS) Xenovulene A verantwortlich. Die ligandfreie Kristallstruktur von AsR6 und die Struktur im Komplex mit Thiolodiphosphat und dem cyclisierten Reaktionsprodukt offenbaren ein neues Farnesyldiphosphat‐Bindungsmotiv. Dieses besteht aus einem binuklearen Mg2+‐Cluster und der essenziellen Aminosäure K289 und es ist in allen an der TS‐Bildung beteiligten Humulen‐Synthasen konserviert. Struktur‐geleitete ortsgerichtete Mutagenese von AsR6 und dem homologen Enzym EupR3 identifiziert eine einzelne Aminosäure, L285/M261, die die Produktion von entweder 2E,6E,9E‐ oder 2Z,6E,9E‐Humulen bestimmt. Ein möglicher Mechanismus für die beobachtete Stereoselektivität wurde mit verschiedenen Isoprenoiden untersucht und die Ergebnisse zeigen, dass M261 die Kontrolle über die Produktbildung hat. [ABSTRACT FROM AUTHOR]

The non‐canonical terpene cyclase AsR6 is responsible for the formation of 2E,6E,9E‐humulene during the biosynthesis of the tropolone sesquiterpenoid (TS) xenovulene A. The structures of unliganded AsR6 and of AsR6 in complex with an in crystallo cyclized reaction product and thiolodiphosphate reveal a new farnesyl diphosphate binding motif that comprises a unique binuclear Mg2+‐cluster and an essential K289 residue that is conserved in all humulene synthases involved in TS formation. Structure‐based site‐directed mutagenesis of AsR6 and its homologue EupR3 identify a single residue, L285/M261, that controls the production of either 2E,6E,9E‐ or 2Z,6E,9E‐humulene. A possible mechanism for the observed stereoselectivity was investigated using different isoprenoid precursors and results demonstrate that M261 has gatekeeping control over product formation. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development. [ABSTRACT FROM AUTHOR]

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/ β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from selfinflicted lysis while ensuring its activity at the pathogen-host interface. [ABSTRACT FROM AUTHOR]

Burkholderia encompasses a group of ubiquitous Gram-negative bacteria that includes numerous saprophytes as well as species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored, redox-active secondary metabolites called phenazines. Phenazines contribute to competitiveness, biofilm formation, and virulence in the opportunistic pathogen Pseudomonas aeruginosa, but knowledge of their diversity, biosynthesis, and biological functions in Burkholderia is lacking. In this study, we screened publicly accessible genome sequence databases and identified phenazine biosynthesis genes in multiple strains of the Burkholderia cepacia complex, some isolates of the B. pseudomallei clade, and the plant pathogen B. glumae. We then focused on B. lata ATCC 17760 to reveal the organization and function of genes involved in the production of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. Using a combination of isogenic mutants and plasmids carrying different segments of the phz locus, we characterized three novel genes involved in the modification of the phenazine tricycle. Our functional studies revealed a connection between the presence and amount of phenazines and the dynamics of biofilm growth in flow cell and static experimental systems but at the same time failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions. [ABSTRACT FROM AUTHOR]

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co‐incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA‐negative mutant in A549 epithelial cells and THP‐1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin‐like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1′ and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination. [ABSTRACT FROM AUTHOR]

Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42. [ABSTRACT FROM AUTHOR]

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNFY, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool. SYNOPSIS: Single‐chain bacterial cytotoxic necrotizing factor (CNF) toxins constitutively activate small Rho GTPases in host cells, leading to actin rearrangements and multinucleation. Here, crystal structures and accompanying cell biological experiments using CNFY from Yersinia pseudotuberculosis reveal the molecular basis of CNFY secretion, host cell binding, and translocation into the host cell cytosol. The CNFY structure reveals five domains, D1–5, with distinct functions in cell entry and RhoA activation.Domains D1–3 constitute a minimal translocation unit that can be employed as broad‐specificity protein delivery tool.A structure of the enzymatically‐active fragment D4–5 reveals structural rearrangements that accompany its release into the host cell cytosol.D4 constitutes a DUF4765 domain and displays structural similarity to ADP‐ribosyl transferases. [ABSTRACT FROM AUTHOR]

Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1. While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1, the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom‐to‐bottom β‐propeller homodimer and confirmed this by multi‐angle light and small‐angle X‐ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives. Database: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and –E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella , RhoB is specifically required for Salmonella -induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella -induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB. • The Salmonella effector SopB induces expression of host cell RhoB. • RhoB expression and SopB secretion enhance each other in a positive feedback loop. • RhoB deficiency results in poor survival of Salmonella. • Salmonella -induced autophagy is abrogated in cells lacking RhoB. [ABSTRACT FROM AUTHOR]

Kinetic target‐guided synthesis represents an efficient hit‐identification strategy, in which the protein assembles its own inhibitors from a pool of complementary building blocks via an irreversible reaction. Herein, we pioneered an in situ Ugi reaction for the identification of novel inhibitors of a model enzyme and binders for an important drug target, namely, the aspartic protease endothiapepsin and the bacterial β‐sliding clamp DnaN, respectively. Highly sensitive mass‐spectrometry methods enabled monitoring of the protein‐templated reaction of four complementary reaction partners, which occurred in a background‐free manner for endothiapepsin or with a clear amplification of two binders in the presence of DnaN. The Ugi products we identified show low micromolar activity on endothiapepsin or moderate affinity for the β‐sliding clamp. We succeeded in expanding the portfolio of chemical reactions and biological targets and demonstrated the efficiency and sensitivity of this approach, which can find application on any drug target. [ABSTRACT FROM AUTHOR]

Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site. [ABSTRACT FROM AUTHOR]

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus. We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus. Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus. We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid. [ABSTRACT FROM AUTHOR]

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes. [ABSTRACT FROM AUTHOR]

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa , making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd 1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d 1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d 1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d 1 -binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d 1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417. Unlabelled Image • Cofactor-free dehydrogenase NirN contains a cyctochrome c and a β-propeller domain. • NirN is similar to nitrite reductase NirS but shows a different domain orientation. • The active site is at the center of the β-propeller and interfaces with the cyt- c domain. • The flexible His323 allows substrate binding, and Tyr461 is required for catalysis. • Electron transfer from substrate but not product to heme c is energetically favored. [ABSTRACT FROM AUTHOR]

The Dispase autolysis‐inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven‐bladed β‐propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP‐mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC‐labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three‐step mechanism defined by (a) fast DAIP–metalloprotease complex formation, (b) slower DAIP‐mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C‐terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis‐prone peptide bonds, suggesting their susceptibility to DAIP‐mediated decomposition, which may offer opportunities for pharmaceutical applications. Databases: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). Enzymes: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25. The Dispase autolysis‐inducing protein (DAIP) from Streptomyces mobaraensis adopts a seven‐bladed β‐propeller fold mediating metalloproteases self‐degradation. The present study now reveals strong attraction of bacillolysin and thermolysin by DAIP, complex formation, and protein twisting followed by fragmentation as indicated by SYPRO orange and FITC‐labeled bacillolysin. The biochemical data are completed by crystallized DAIP in complex with a C‐terminal thermolysin fragment. [ABSTRACT FROM AUTHOR]