Structural characterization of antibiotic self-immunity tRNA synthetase in plant tumour biocontrol agent

Antibiotic-producing microbes evolved self-resistance mechanisms to avoid suicide. The biocontrol Agrobacterium radiobacter K84 secretes the Trojan Horse antibiotic agrocin 84 that is selectively transported into the plant pathogen A. tumefaciens and processed into the toxin TM84. We previously showed that TM84 employs a unique tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase (LeuRS), while the TM84-producer prevents self-poisoning by expressing a resistant LeuRS AgnB2. We now identify a mechanism by which the antibiotic-producing microbe resists its own toxin. Using a combination of structural, biochemical and biophysical approaches, we show that AgnB2 evolved structural changes so as to resist the antibiotic by eliminating the tRNA-dependence of TM84 binding. Mutagenesis of key resistance determinants results in mutants adopting an antibiotic-sensitive phenotype. This study illuminates the evolution of resistance in self-immunity genes and provides mechanistic insights into a fascinating tRNA-dependent antibiotic with applications for the development of anti-infectives and the prevention of biocontrol emasculation

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Development and evaluation of a screening system for the molecular evolution of the steroid-15beta-hydroxylase (CYP106A2) from Bacillus megaterium ATCC 13368

Steroide spielen eine bedeutende Rolle als Arzneimittel. Die Produktion der Steroide stellt eine besondere Herausforderung dar, da sie spezifische Reaktionen am Steroidkörper erfordern. Dabei wird insbesondere für Hydroxylierungsreaktionen auf Mikroorganismen bzw. mikrobielle Enzyme zurückgegriffen, da diese die Hydroxylierungen im Gegensatz zu chemisch katalysierten Reaktionen äußerst regio- und stereospezifisch durchführen. Die mikrobiellen Enzyme sind z. T. gut an die Anforderungen des Einsatzes in der Produktion angepasst. Oftmals müssen die Mikroorganismen oder die Enzyme aber erst adaptiert werden. Das Ziel dieser Arbeit war die Etablierung und Evaluierung eines Screeningsystems für die gerichtete Evolution eines Steroid-hydroxylierenden Enzyms, der 15beta-Hydroxylase (CYP106A2) aus Bacillus megaterium ATCC 13368. Dieses lösliche bakterielle Cytochrom P450 ist eines der wenigen bekannten und besser charakterisierten Steroid-hydroxylierenden Cytochrome P450, womit es ein hohes Potenzial für eine biotechnologische Nutzung hat. Durch die gerichtete Evolution wurden Mutanten mit veränderter Stereo- und Regioselektivität der Hydroxylierung sowie erhöhten Hydroxylierungsaktivitäten gewonnen.Steroids play an important role as drugs. The production of the steroids is difficult due to the necessity of specific modifications on the steroid structure. Therefore, the activity of microorganisms or their enzymes is used to perform especially hydroxylation reactions. These enzymes catalyze the reactions with a high regio- and stereoselectivity. The enzymes are sometimes quite well adapted on the requirements of the industrial use. But often they have to become adapted on these requirements. The aim of this work was the development and the evaluation of a screening system for the directed evolution of a steroid-hydroxylating enzyme, the 15beta-hydroxylase (CYP106A2) from Bacillus megaterium ATCC 13368. This soluble bacterial enzyme is one of the few known and better characterized steroid-hydroxylating cytochromes P450. Therefore, it has a high potential for the use in biotechnological processes. Mutants with altered stereo- and regioselectivity of hydroxylation and increased hydroxylation activity are generated by the use of the directed evolution

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

9 pags, 5 figs, 2 tabsLeucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA Leu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3′-end. Our findings reveal an intriguing tRNA-dependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design. © 2013 Macmillan Publishers Limited. All rights reserved

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens

International audiencePeriplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs

A simplified schematic representing the agrocinopine A and agrocin 84 roles in <i>A</i>. <i>tumefaciens</i> C58.

<p>Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain <i>A</i>. <i>radiobacter</i> K84 (which colonizes the same plant environment as the <i>A</i>. <i>tumefaciens</i> C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of <i>A</i>. <i>tumefaciens</i> C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In <i>A</i>. <i>tumefaciens</i> C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing <i>acc</i> and <i>traR</i> genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the <i>tra</i>, <i>trb</i> and <i>rep</i> genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.</p

Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-<i>O</i>-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.

<p>Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-<i>O</i>-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.</p

Synthesis scheme.

<p>(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.</p

Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.

<p>Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.</p