Wulf Blankenfeldt

Helmholtz Centre for Infection Research | HZI · Research Group of Structure and Function of Proteins

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridi...

In the search for new antitubercular compounds, we leveraged target-directed dynamic combinatorial chemistry (tdDCC) as an efficient hit-identification method. In tdDCC, the target selects its own binders from a dynamic library generated in situ, reducing the number of compounds that require synthesis and evaluation. We combined a total of twelve h...

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in Bacillaceae, employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based ide...

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselecti...

Although ethers are common in secondary natural products, they are an underrepresented functional group in primary metabolism. As such, there are comparably few enzymes capable of constructing ether bonds in a general fashion. However, such enzymes are highly sought after for synthetic applications as they typically operate with higher regioselecti...

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2 S,4 R)−4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from...

Background Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known as a major receptor for oxidized low-density lipoproteins (oxLDL) and plays a significant role in the genesis of atherosclerosis. Recent research has shown its involvement in cancer, ischemic stroke, and diabetes. LOX-1 is a C-type lectin receptor and is involved in...

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 Pl...

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile . D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostrid...

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...

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...

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2S,4R)-4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from t...

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pat...

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 P...

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction d...

Quorum Sensing Inhibitor In article number 2204443, Martin Empting and colleagues describe the medicinal‐chemistry‐driven discovery of a new quorum sensing inhibitor (QSI) against Pseudomonas aeruginosa. The discovered QSI synergized with standard‐of‐care antibiotic tobramycin in a murine infection model providing an in vivo proof‐of‐concept.

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...

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa , with the rhl circuit playing the leading role in late and chronic infections. The majority of traits contro...

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activatio...

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp...

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits control...

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 inte...

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 inte...

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....

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...

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 co...

A short and divergent route towards new derivatives of 2-(trifluoromethyl)pyridines as potent inverse agonists of the bacterial target PqsR against Pseudomonas aeruginosa (PA) infections is described. This Gram-negative pathogen causes severe nosocomial infections and common antibiotic treatment options are rendered ineffective due to resistance is...

In article number 2004369, Martin Empting and colleagues describe a new generation of dual‐loaded nanoparticles enabling efficient eradication of Pseudomonas aeruginosa biofilms. The squalene‐based nanocarrier facilitates co‐delivery of a novel pathogenspecific Quorum Sensing Inhibitor (QSI) together with the standard‐of‐care antibiotic tobramycin....

Significance Secreted bacterial phospholipases, which hydrolyze host membranes, may also harm the pathogen itself. Sophisticated activation mechanisms are therefore required to prevent self-damage before protein export but warrant activity at the pathogen–host interface. Here we structurally analyzed PlaB, a surface-exposed highly active phospholip...

AsR6 ist eine Klasse-I-Sesquiterpencyclase mit einzigartiger Bi-Magnesium Diphosphat-Bindungsstelle und es fehlen die kanonischen konservierten aspartatreichen Signatursequenzen. Ein Magnesium wird durch das ϵ-Ammonium-Ion eines konservierten Lysins ersetzt. AsR6 kann FPP und beide Enantiomere von NPP als Substrat verwenden. Außerdem steuert eine e...

Burkholderia encompass a group of ubiquitous Gram-negative bacteria that include 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 forma...

Antimicrobial resistance is a global threat that kills at least 75,000 people every year worldwide and causes extended hospital stays. In the coming 10 years, antimicrobial resistance is projected to have huge health and economic burden on countries, and the scarcity of available antibiotics further worsens the situation. Antimicrobial resistance r...

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...

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 t...

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished...

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-dim...

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 s...

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, respe...

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio‐ and stereoselectivity at the 16α‐position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, con...

The Ugi four‐component reaction (Ugi‐4CR) was employed as a new reaction in kinetic target‐guided synthesis (KTGS). Applying the reaction on the bacterial β sliding clamp (DnaN) (middle) resulted in marked acceleration of product assembly from a library of eight building blocks representing four chemical classes. UPLC‐HRMS analysis of the in situ U...

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...

The virulence factor and phospholipase PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for en...

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...

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 unco...

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...

Cytotoxic necrotizing factors (CNFs) are single-chain exotoxins. They are secreted by several bacterial pathogens to modulate cytokinetic/oncogenic and inflammatory processes through activation host cell Rho-GTPases, but their secretion-translocation mechanism still remains an enigma. Here, we determined the crystal structure of full-length Yersini...

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 refer...

Ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyzes electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a three-component cytochrome P450 monooxygenase system in T. fusca. Here, FdR9 was overexpressed and purified...

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of pro...

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 referre...

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by cd 1 nitrite reductase NirS in e.g. 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 isobact...

Kinetic target-guided synthesis represents an efficient hit-identification strategy, in which the protein assembles its own inhibitors from a pool of 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 Front Cover illustrates key steps of a successful fragment‐growing campaign starting from an enthalpy‐driven, high‐affinity fragment identified via biophysical methods against bacterial quorum sensing receptor PqsR. This transcription regulator is an attractive drug target for the design of so‐called pathoblockers against Pseudomonas aeruginosa...

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 e.g. to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infectio...

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 to molybdopterin (MPT) in the second p...

Hit‐to‐lead optimization is a critical phase in drug discovery. Herein, we report on the fragment‐based discovery and optimization of 2‐aminopyridine derivatives as a novel lead‐like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomo...

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 p...

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 utilizat...

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 tha...

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 h...

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 tha...

HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability,...

Video S4. Migration Patterns of Dictyostelium Cells Lacking or Harboring Distinct WRCs, Related to Figure 2 Pseudopod formation in wild type parental strain Ax3, Pir121 knock out and cells expressing wild type and mutant (A and D site) Pir121-EGFP. Cells were imaged every 3 s, and time-lapse movie is shown at 10 frames/second.

Video S3. Compromised Protrusion with WRC Harboring the D Site Mutant of Sra-1, Related to Figure 3 High magnification, phase contrast video microscopy of individual Sra-1/PIR121 double KO B16-F1 melanoma cells (clone #3) transfected with EGFP-tagged versions (not shown) of wild type Sra-1 (WT), the D site mutant (Y967A) or the latter additionally...

Video S1. Reduction of B16-F1 Cell Migration upon Sra-1/PIR121 Ablation, Related to Figure 1 Phase contrast, time-lapse microscopy of B16-F1 wildtype, Sra-1/PIR121 KO clone #3 and the latter re-expressing EGFP-tagged Sra-1, as indicated, for restoration of lamellipodia-dependent migration. Time is in hours and minutes, scale bar is valid for all p...

Video S2. Confocal Imaging of Dictyostelium Cells Migrating under Agarose, Related to Figure 2 AiryScan imaging of cells to visualize WRC complex (green) and Arp2/3 complex (red) localization in wild type and PIR121 null cells expressing wild type and mutant (A or D site) Pir121-EGFP. Cells were imaged every 2 s, and resulting time-lapse movie is...

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/...

Inteins remove themselves out of a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance tow...

Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH anti...

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 c...

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 non-ribosomal peptide synthetases which produce a polype...

Cell migration often involves the formation of sheet‐like lamellipodia, generated by actin filament branching through Arp2/3 complex. In these structures, the latter is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family. Recent structural studies defined two independent Rac1 binding sites on WRC within the Sra‐...

Pseudomonas aeruginosa is a bacterial pathogen that causes life‐threatening infections in immunocompromised patients. It produces a large armory of saturated and mono‐unsaturated 2‐alkyl‐4(1H)‐quinolones (AQs) or AQ N‐oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors, are involved in membrane vesicle fo...

The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco- dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP to cyclic pyranopterin monophoshpate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP) and finally...

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in erg...

Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the con...

Oxygen-containing heterocycles are a common structural motif of polyketide natural products and contribute significantly to their biological activity. However, there are only limited reports of the responsible cyclases. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratas...

Oxygen-containing heterocycles are a common structural motif of polyketide natural products and contribute significantly to their biological activity. However, there are only limited reports of the responsible cyclases. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratas...

Halohydrin dehalogenases (HHDHs) are of biotechnological interest due to their promiscuous epoxide ring-opening activity with a set of negatively charged nucleophiles, enabling the formation of C–C, C–N, or C–O bonds. The recent discovery of HHDH-specific sequence motifs aided the identification of a large number of halohydrin dehalogenases from pu...

In vitro reconstitution and biochemical analysis of natural product biosynthetic pathways remains a challenging endeavor, especially if megaenzymes of the nonribosomal peptide synthetase (NRPS) type are involved. In theory, all biosynthetic steps may be deciphered using mass spectrometry (MS)-based analyses of both the carrier protein-coupled inter...