Gottfried J. Palm

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, Mpro, a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus Mpro is reported. The structure was refined to 1.96 Å resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that Mpro self‐processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin‐like fold that is connected by a 16 residue loop to an extra domain featuring a novel α‐helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of Mpro substrates. Interactions involving the N‐terminus and the α‐helical domain stabilize the loop in the orientation required for trans‐cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Escherichia coli asparaginase II catalyzes the hydrolysis of l‐asparagine to l‐aspartate via a threonine‐bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr‐89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild‐type enzyme, but an aspartyl moiety is covalently bound to Thr‐12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.

Spectral variants of green fluorescent protein.

Publisher Summary The use of green fluorescent protein (GFP) for many applications in biological sciences strongly depends on its spectral properties. GFP originates from the jellyfish Aequorea victoria, in which its fluorescence serves to convert the blue light produced by aequorin to green light. Purified protein that retains fluorescence has been the object of intense investigations for many years. Denaturation by harsh treatment, however, can abolish fluorescence and change the absorption spectrum of GFP. The spectrum of the native protein is also affected by many other parameters, such as mutations, protein concentration, ionic strength, temperature, and pH. However, the stability of GFP in a wide pH range (pH 5-12), at high temperature (T m = 78°), in chaotropic reagents (8 M urea), and against proteolysis makes it attractive for practical use. Expression has been optimized by mutations that change the promoter, ribosome binding site, or codon usage; eliminate splicing sites; or enhance folding. Even though such procedures can drastically increase the overall fluorescence by GFP per cell, as the spectra of purified GFP remain the same in all these cases. Numerous mutations in and around the chromophore, however, do change the spectra. Taken together, these advances enable the use of GFP for detection of promoter activity and for localization of fusion proteins. New applications such as simultaneous localization of two fusion proteins and fluorescence resonance energy transfer (FRET) for detection of Ca 2+ concentration or proteolytic activity have become possible through the use of GFP mutants of different colors.

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 pKa 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.

Structural basis for a new tetracycline resistance mechanism relying on the TetX monooxygenase

The flavin‐dependent monooxygenase TetX confers resistance to all clinically relevant tetracyclines, including the recently approved, broad‐spectrum antibiotic tigecycline (Tygacil®) which is a critical last‐ditch defense against multidrug‐resistant pathogens. TetX represents the first resistance mechanism against tigecycline, which circumvents both the tet‐gene encoded resistances, relying on active efflux of tetracyclines, and ribosomal protection proteins. The alternative enzyme‐based mechanism of TetX depends on regioselective hydroxylation of tetracycline antibiotics to 11a‐hydroxy‐tetracyclines. Here, we report the X‐ray crystallographic structure determinations at 2.1 Å resolution of native TetX from Bacteroides thetaiotaomicron and its complexes with tetracyclines. Our crystal structures explain the extremely versatile substrate diversity of the enzyme and provide a first step towards the rational design of novel tetracycline derivatives to counter TetX‐based resistance prior to emerging clinical observations.

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.

Crystallization, structure solution and refinement of hen egg-white lysozyme at pH 8.0 in the presence of MPD.

Hen egg-white lysozyme has been crystallized at slightly alkaline pH using 2-methyl-2,4-pentanediol (MPD) as the precipitant. The crystals are nearly isomorphous to crystals grown at acidic pH using sodium chloride as the precipitant. However, the growth kinetics differ markedly between the two conditions. The major reason for this is a molecule of MPD that binds tightly in between two lysozyme molecules and favors the growth of the crystals along the crystallographic c direction over growth perpendicular to it.

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.

Nonantibiotic properties of tetracyclines: structural basis for inhibition of secretory phospholipase A2.

Secretory phospholipase A(2) is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A(2), which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 A resolution of the minoTc complex of phospholipase A(2) (PLA(2)) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca(2+)-binding loop, preventing Ca(2)(+) binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA(2) is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA(2) was determined by surface plasmon resonance, resulting in a dissociation constant K(d)=1.8 x 10(-)(4) M.

Dielectric barrier discharge plasma treatment on E. coli: Influence of CH4/N2, O2, N2/O2, N2, and Ar gases

Atmospheric pressure dielectric barrier discharge (DBD) plasma has been employed on Gram-negative bacteria, Escherichia coli BL21. Treatment was carried out using plasma generated with different compositions of gases: CH4/N2 (1:2), O2, N2/O2 (1:1), N2, and Ar, and by varying plasma power and treatment time. E. coli cells were exposed under the DBD plasma in triplicates, and their surviving numbers were observed in terms of colony forming units. It has been observed that the CH4/N2 plasma exhibits relatively higher sterilization property toward E. coli compared to plasma generated by using O2, N2/O2, N2, and Ar gas mixtures. The time to kill up to 90% of the initial population of the E. coli cells was found to be about 2–3 min for CH4/N2 and O2 gas mixture DBD plasma. A prolongation of treatment time and an increase in the dissipated power significantly improved the E. coli killing efficiency of the atmospheric pressure DBD plasma.