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The nematode mutualistic bacterium Photorhabdus asymbiotica produces a large virulence-associated multifunctional protein toxin named PaTox. A glycosyltransferase domain and a deamidase domain of this large toxin function as effectors that specifically target host Rho GTPases and heterotrimeric G proteins, respectively. Modification of these intracellular regulators results in toxicity toward insects and mammalian cells. In this study, we identified a cysteine protease–like domain spanning PaTox residues 1844–2114 (PaToxP), upstream of these two effector domains and characterized by three conserved amino acid residues (Cys-1865, His-1955, and Asp-1975). We determined the crystal structure of the PaToxP C1865A variant by native single-wavelength anomalous diffraction of sulfur atoms (sulfur-SAD). At 2.0 Å resolution, this structure revealed a catalytic site typical for papain-like cysteine proteases, comprising a catalytic triad, oxyanion hole, and typical secondary structural elements. The PaToxP structure had highest similarity to that of the AvrPphB protease from Pseudomonas syringae classified as a C58-protease. Furthermore, we observed that PaToxP shares structural homology also with non–C58-cysteine proteases, deubiquitinases, and deamidases. Upon delivery into insect larvae, PaToxP alone without full-length PaTox had no toxic effects. Yet, PaToxP expression in mammalian cells was toxic and enhanced the apoptotic phenotype induced by PaTox in HeLa cells. We propose that PaToxP is a C58-like cysteine protease module that is essential for full PaTox activity.

The Gram-negative bacterium Aeromonas salmonicida is a fish pathogen for various fish species worldwide. Aeromonas salmonicida subsp. achromogenes produces the extracellular, toxic zinc endopeptidase AsaP1. Crystal structure analyses at 2.0 Å resolution of two proteolytically inactive AsaP1 variants show the polypeptide folding of the protease domain and the propeptide domain. These first crystal structure analyses of a precursor of a deuterolysin-like aspzincin protease provide insights into propeptide function, and specific substrate binding. A lysine side chain of the propeptide binds in the hydrophobic S1'-pocket interacting with three carboxylate side chains. An AsaP1 variant with a lysine to alanine exchange identifies the chaperone function of the propeptide.

A new bacterial toxin from pathogen Photorhabdus asymbiotica is now described. The toxin contains a domain with glycosyltransferase activity that modifies a tyrosine residue of Rho GTPases in the host cell, inhibiting Rho activation and interaction with downstream effectors. The second domain is a glutamine deamidase that blocks GTP hydrolysis by Gaq/11 and Gai proteins. Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP–N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-A resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. 1H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

It is well known that protein crystallization is affected by several different parameters such as the composition of the reservoir solution, the protein concentration, the pH and the temperature. An effect of different temperatures during setup of crystallization experiments was observed for a metalloendopeptidase (AsaP1(E294A)). Spontaneous protein precipitation was reduced and the crystal shape could be improved by decreasing the temperature during crystallization setup.

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2–4). To explain these unique properties, its crystal structure has been determined at 2.0 A resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The Km and kcat values for 1-bromobutane were 24.0 mM and 8.08 s−1. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences., We thank the German Research Foundation (DFG, Grant Bo1862/4-1) for their financial support.

DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2(1)2(1)2. The DppA crystal diffracted X-rays to 1.9 A resolution using an in-house X-ray generator.

Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P2(1)2(1)2(1)) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 A, beta = 109.3 degrees for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 A, beta = 112.4 degrees for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 A. The crystals of the two different mutants diffracted X-rays beyond 2.0 A resolution.