Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

Structural and Biophysical Insights into SPINK1 Bound to Human Cationic Trypsin.

(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1-TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.

Structural Basis for (2R,3R)-Taxifolin Binding and Reaction Products to the Bacterial Chalcone Isomerase of Eubacterium ramulus.

The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by a reverse Michael addition. The overall fold and the constitution of the active site of the enzyme completely differ from the well-characterised chalcone isomerase of plants. For (+)-taxifolin, CHI catalyses the intramolecular ring contraction to alphitonin. In this study, Fwe perform crystal structure analyses of CHI and its active site mutant His33Ala in the presence of the substrate taxifolin at 2.15 and 2.8 Å resolution, respectively. The inactive enzyme binds the substrate (+)-taxifolin as well defined, whereas the electron density maps of the native CHI show a superposition of substrate, product alphitonin, and most probably also the reaction intermediate taxifolin chalcone. Evidently, His33 mediates the stereospecific acid-base reaction by abstracting a proton from the flavonoid scaffold. The stereospecificity of the product is discussed.

Discovery and Design of Family†VIII Carboxylesterases as Highly Efficient Acyltransferases.

Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".

Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases.

Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.

Mapping the recognition domains of pneumococcal fibronectin-binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats.

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin-binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non-human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both ß-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1 , FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase.

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.

Crystallographic characterization of the (R)-selective amine transaminase from Aspergillus fumigatus.

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R)-selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Šresolution. The refined structure at 1.27 Šresolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and D-amino-acid aminotransferases.

Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase.

Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction.

Bacteria employ lysine acetylation of transcriptional regulators to adapt gene expression to cellular metabolism.

The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD+-dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD+ and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.

Putative dioxygen-binding sites and recognition of tigecycline and minocycline in the tetracycline-degrading monooxygenase TetX.

Expression of the aromatic hydroxylase TetX under aerobic conditions confers bacterial resistance against tetracycline antibiotics. Hydroxylation inactivates and degrades tetracyclines, preventing inhibition of the prokaryotic ribosome. X-ray crystal structure analyses of TetX in complex with the second-generation and third-generation tetracyclines minocycline and tigecycline at 2.18 and 2.30 Šresolution, respectively, explain why both clinically potent antibiotics are suitable substrates. Both tetracyclines bind in a large tunnel-shaped active site in close contact to the cofactor FAD, pre-oriented for regioselective hydroxylation to 11a-hydroxytetracyclines. The characteristic bulky 9-tert-butylglycylamido substituent of tigecycline is solvent-exposed and does not interfere with TetX binding. In the TetX-minocycline complex a second binding site for a minocycline dimer is observed close to the active-site entrance. The pocket is formed by the crystal packing arrangement on the surface of two neighbouring TetX monomers. Crystal structure analysis at 2.73 Šresolution of xenon-pressurized TetX identified two adjacent Xe-binding sites. These putative dioxygen-binding cavities are located in the substrate-binding domain next to the active site. Molecular-dynamics simulations were performed in order to characterize dioxygen-diffusion pathways to FADH2 at the active site.

Crystallization and preliminary X-ray diffraction studies of the (R)-selective amine transaminase from Aspergillus fumigatus.

The (R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C222(1). X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing.

Crystal structures of free and ligand-bound forms of the TetR/AcrR-like regulator SCO3201 from Streptomyces coelicolor suggest a novel allosteric mechanism.

TetR/AcrR-like transcription regulators enable bacteria to sense a wide variety of chemical compounds and to dynamically adapt the expression levels of specific genes in response to changing growth conditions. Here, we describe the structural characterisation of SCO3201, an atypical TetR/AcrR family member from Streptomyces coelicolor that strongly represses antibiotic production and morphological development under conditions of overexpression. We present crystal structures of SCO3201 in its ligand-free state as well as in complex with an unknown inducer, potentially a polyamine. In the ligand-free state, the DNA-binding domains of the SCO3201 dimer are held together in an unusually compact conformation and, as a result, the regulator cannot span the distance between the two half-sites of its operator. Interaction with the ligand coincides with a major structural rearrangement and partial conversion of the so-called hinge helix (α4) to a 310 -conformation, markedly increasing the distance between the DNA-binding domains. In sharp contrast to what was observed for other TetR/AcrR-like regulators, the increased interdomain distance might facilitate rather than abrogate interaction of the dimer with the operator. Such a 'reverse' induction mechanism could expand the regulatory repertoire of the TetR/AcrR family and may explain the dramatic impact of SCO3201 overexpression on the ability of S. coelicolor to generate antibiotics and sporulate.

A FRET enzyme-based probe for monitoring hydrogen sulfide.

Fluorescently labeled cobalt peptide deformylase (Co-PDF) can be efficiently used as a fluorescence-resonance-energy-transfer-based sensing device for hydrogen sulfide (H(2)S). The proof of concept of our sensor system is substantiated by spectroscopic, structural, and theoretical results. Monohydrogen sulfide coordination to Co-PDF and Ni-PDF was verified by X-ray crystallography. Density functional theory calculations were performed to gain insight into the characteristics of the coordination adduct between H(2)S and the cobalt cofactor in Co-PDF.

Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase.

Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Šin the trigonal space group P3(2)21, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known.

Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin.

Objective: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). This ratio is reversed during chronic alcohol abuse, pancreatic cancer, or pancreatitis due to selectively upregulated expression of TRY2, causing anionic trypsin to become the predominant isoform. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity. Methods: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1-TRY1 model. Results: All trypsin isoforms display similar kinetic properties. The crystal structure of TRY2 reveals that the enzyme crystallized in the autoproteolytic state with Arg122 placed in the S1 binding pocket and the corresponding loop cleaved. The TRY2-TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface. Conclusion: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated.

The induction mechanism of the flavonoid-responsive regulator FrrA.

Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation-promoting genetic programme in the presence of host-produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoid-responsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR-like fold whose DNA-binding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C-H contacts to the π-system of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA-binding domain and the absence of structural change upon ligand binding are corroborated by small-angle X-ray scattering (SAXS) experiments in solution. Together with a model of the promoter-bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA-binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.