A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities.

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein-protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.

A Rationally Designed Supercharged Protein-Enzyme Chimera Self-Assembles In Situ to Yield Bifunctional Composite Textiles.

Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S-)-mediated self-assembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number (kcat). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S-, driving self-assembly into [scGFP-arPTE][S-] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S-] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center.

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as "minimized" or "expanded" based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens transfer DNA (T-DNA) and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1 to VirB11 and VirD4. We visualized the pKM101-encoded T4SS in its native cellular context by in situ cryo-electron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by in situ CryoET but differs strikingly from the IMC of the purified T4SSR388, whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers of dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer. IMPORTANCE Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryo-electron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely related T4SS analyzed in vitro by single-particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for the development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

A Possible Mechanism of Graphene Oxide to Enhance Thermostability of D-Psicose 3-Epimerase Revealed by Molecular Dynamics Simulations.

Thermal stability is a limiting factor for effective application of D-psicose 3-epimerase (DPEase) enzyme. Recently, it was reported that the thermal stability of DPEase was improved by immobilizing enzymes on graphene oxide (GO) nanoparticles. However, the detailed mechanism is not known. In this study, we investigated interaction details between GO and DPEase by performing molecular dynamics (MD) simulations. The results indicated that the domain (K248 to D268) of DPEase was an important anchor for immobilizing DPEase on GO surface. Moreover, the strong interactions between DPEase and GO can prevent loop α1'-α1 and ß4-α4 of DPEase from the drastic fluctuation. Since these two loops contained active site residues, the geometry of the active pocket of the enzyme remained stable at high temperature after the DPEase was immobilized by GO, which facilitated efficient catalytic activity of the enzyme. Our research provided a detailed mechanism for the interaction between GO and DPEase at the nano-biology interface.

An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33.

Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical pathway have been identified and characterized, except for the one catalyzing the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. Based on a previous genomic and transcriptomic analysis, an open reading frame (ORF) annotated to encode aldehyde dehydrogenase (Ald) in the nicotine-degrading cluster was predicted to be responsible for this step. In this study, we heterologously expressed the enzyme and identified its function by biochemical assay and mass spectrum analysis. It was found that Ald catalyzes the NAD-specific dehydrogenation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. With the nonhydroxylated analog 3-succinoyl-semialdehyde-pyridine (SAP) as a substrate, Ald had a specific activity of 10.05 U/mg at pH 9.0 and apparent Km values of around 58.68 µM and 0.41 mM for SAP and NAD+, respectively. Induction at low temperature and purification and storage in low-salt buffers were helpful to prevent its aggregation and precipitation. Disruption of the ald gene caused a lower growth rate and biomass of strain S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine. Ald has a broad range of substrates, including benzaldehyde, furfural, and acetaldehyde. Recombinant Escherichia coli cells harboring the ald gene can efficiently convert furfural to 2-furoic acid at a specific rate of 0.032 mmol min-1 g dry cells-1, extending the application of Ald in the catalysis of bio-based furan compounds. These findings provide new insights into the biochemical mechanism of the nicotine-degrading hybrid pathway and the possible application of Ald in industrial biocatalysis. IMPORTANCE Nicotine is one of the major toxic N-heterocyclic aromatic alkaloids produced in tobacco plants. Manufacturing tobacco and smoking may lead to some environmental and public health problems. Microorganisms can degrade nicotine by various biochemical pathways, but the biochemical mechanism for nicotine degradation has not been fully elucidated. In this study, we identified an aldehyde dehydrogenase responsible for the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine; this was the only uncharacterized enzyme in the hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33. Similar to the known aldehyde dehydrogenase, the NAD-specific homodimeric enzyme presents a broad substrate range with high activity in alkaline and low-salt-containing buffers. It can catalyze not only the aldehyde from nicotine degradation but also those of benzaldehyde, furfural, and acetaldehyde. It was found that recombinant Escherichia coli cells harboring the ald gene could efficiently convert furfural to valuable 2-furoic acid, demonstrating its potential application for enzymatic catalysis.

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily.

Aconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

Single stranded DNA annealing is a conserved activity of telomere resolvases.

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA's annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

A YoeB toxin cleaves both RNA and DNA.

Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

Immobilization of Agrobacterium tumefaciensd-psicose 3-epimerase onto titanium dioxide for bioconversion of rare sugar.

d-Psicose (d-ribo-2-hexulose or d-allulose) is the Carbon-3 epimer of d-fructose sugar and considered as an unnatural (rare) sugar found in low amount in nature. It has about 70% of the relative sweetness but 0.3% of the energy of sucrose, which is suggested as the most suitable sucrose substitute for food additives. Enzymatic biosynthesis using ketose 3-epimerases is a necessary procedure for the production of d-Psicose from d-fructose. However, significant drawbacks in the application of ketose 3-epimerases at industrial scale observe lower thermal stability as well as bioconversion efficiency, reusability and recovery of the enzyme. We have attempted immobilization of ketose 3-epimerases from Agrobacterium tumefaciens (agtu) d-psicose 3-epimerase (DPEase) on titanium dioxide. Further, Scanning electron microscopy (SEM), inverted microscopy, Fourier transform infrared spectroscopy (FTIR) and UV-vis spectroscopy showed that the enzyme was successfully immobilized on the titanium dioxide (TiO2) surface. Titanium dioxide immobilized agtu-DPEase (TiO2-agtu-DPEase) shows pH optima at 6.0 and 60 °C as a higher working temperature. TiO2-agtu-DPEase showed a half-life of 180 min at 60 °C, which is higher as compared to Agrobacterium tumefaciens (agtu) DPEase (3.99 min at 50 °C). At equilibrium, 36:64 (D-psicose: d-fructose), the bioconversion efficiency was accounted for titanium dioxide immobilized DPEase, which is higher than the agtu-DPEase. Titanium dioxide immobilized DPEase showed bioconversion efficiency up to 9 cycles of reusability.

Enzyme immobilization on glass fiber membrane for detection of halogenated compounds.

Enzyme immobilization using inorganic membranes has enticed increased attention as they not only improve enzyme stability, but also furnish user-friendly biodevices that can be tailored to different applications. Herein, we explored the suitability of the glass fiber membrane for enzyme immobilization and its application for halocarbon detection. For this, halohydrin dehalogenase (HheC) and bovine serum albumin were crosslinked and immobilized on a glass fiber membrane without membrane functionalization. Immobilized HheC exhibited higher storage stability than its free counterpart over 60 days at 4 °C (67% immobilized vs. 8.1% free) and 30 °C (77% immobilized vs. 57% free). Similarly, the thermal endurance of the immobilized HheC was significantly improved. The practical utility of the membrane-immobilized enzyme was demonstrated by colorimetric detection of 1,3-dichloro-2-propanol (1,3-DCP) and 2,3-dibromo-1-propanol (2,3-DBP) as model analytes. Under optimized conditions, the detection limits of 0.06 mM and 0.09 mM were achieved for 1,3-DCP and 2,3-DBP, respectively. The satisfactory recoveries were observed with spiked river and lake water samples, which demonstrate the application potential of immobilized HheC for screening contaminants in water samples. Our results revealed that the proposed frugal and facile approach could be useful for enzyme stabilization, and mitigation of halocarbon pollution.

Quorum quenching acylase impacts the viability and morphological change of Agrobacterium tumefaciens cells.

Acylase is known as a quorum quenching enzyme that degrades N-acyl-homoserine lactones (AHLs), a key signaling molecule in a quorum sensing (QS) mechanism. Acylase I cleaves the acyl-chain in the chemical structures of AHLs, thereby exerting an anti-biofilm effect by the inhibition of bacterial cell-cell communication and resultant secretion of extracellular polymeric substances (EPS). However, the physical and physiological impacts of acylase on bacterial cells remain to be systematically elucidated. This study, therefore, investigated the effect of active and inactive acylase addition on the growth, viability, and cell morphologies of Agrobacterium tumefaciens. For comparison, active and inactive lysozymes were taken as positive controls. The results showed that active acylase inhibited A. tumefaciens cell growth at concentrations ranging from 0.1 to 1000 µg mL-1, and so did active lysozyme. Fluorescent detection by Live/Dead staining underpinned that cell viability of A. tumefaciens decreased at concentrations higher than 0.1 µg mL-1 for both acylase and lysozyme, although lysozyme inflicted higher degree of cellular damage. Moreover, atomic force microscopy unraveled a noticeable distortion of A. tumefaciens cells by both acylase and lysozyme. Together, the results showed that acylase not only blocked AHLs-based QS mechanisms but also compromised cell viability and altered surface morphology of A. tumefaciens cells, as observed by the addition of hydrolase.

On the simultaneous activation of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by pyruvate and fructose 6-phosphate.

Bacterial ADP-glucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. Pyruvate (Pyr) and fructose 6-phosphate (Fru6P) activate the enzyme from Agrobacterium tumefaciens by increasing Vmax about 10- and 20-fold, respectively. Here, we studied the combined effect of both metabolites on the enzyme activation. Our results support a model in which there is a synergistic binding of these two activators to two distinct sites and that each activator leads the enzyme to distinct active forms with different properties. In presence of both activators, Pyr had a catalytically dominant effect over Fru6P determining the active conformational state. By mutagenesis we obtained enzyme variants still sensitive to Pyr activation, but in which the allosteric signal by Fru6P was disrupted. This indicated that the activation mechanism for each effector was not the same. The ability for this enzyme to have more than one allosteric activator site, active forms, and allosteric signaling mechanisms is critical to expand the evolvability of its regulation. These synergistic interactions between allosteric activators may represent a feature in other allosteric enzymes.

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction.

This paper reports a versatile microfluidic chip developed for on-chip crystallization of proteins through the dialysis method and in situ X-ray diffraction experiments. A microfabrication process enabling the integration of regenerated cellulose dialysis membranes between two layers of the microchip is thoroughly described. We also describe a rational approach for optimizing on-chip protein crystallization via chemical composition and temperature control, allowing the crystal size, number and quality to be tailored. Combining optically transparent microfluidics and dialysis provides both precise control over the experiment and reversible exploration of the crystallization conditions. In addition, the materials composing the microfluidic chip were tested for their transparency to X-rays in order to assess their compatibility for in situ diffraction data collection. Background scattering was evaluated using a synchrotron X-ray source and the background noise generated by our microfluidic device was compared to that produced by commercial crystallization plates used for diffraction experiments at room temperature. Once crystals of 3 model proteins (lysozyme, IspE, and insulin) were grown on-chip, the microchip was mounted onto the beamline and partial diffraction data sets were collected in situ from several isomorphous crystals and were merged to a complete data set for structure determination. We therefore propose a robust and inexpensive way to fabricate microchips that cover the whole pipeline from crystal growth to the beam and does not require any handling of the protein crystals prior to the diffraction experiment, allowing the collection of crystallographic data at room temperature for solving the three-dimensional structure of the proteins under study. The results presented here allow serial crystallography experiments on synchrotrons and X-ray lasers under dynamically controllable sample conditions to be observed using the developed microchips.

Simple Purification and Immobilization of His-Tagged Organophosphohydrolase from Cell Culture Supernatant by Metal Organic Frameworks for Degradation of Organophosphorus Pesticides.

Coordinating unsaturated metal sites (CUS) on the surface of metal-organic frameworks (MOFs) could be used to adsorb His-tagged proteins. The specific adsorption between CUS and His-tagged proteins could reduce preparation steps, shorten preparation time, and could also avoid the binding between the metal ion of metalloenzyme active center and the chelating agent to ensure the enzyme activity. In this study, MIL-88A was synthesized by hydrothermal method and used to purify and immobilize His-tagged organophosphohydrolase (OpdA) in one step for organophosphate bioremediation. Under optimized conditions, OpdA@MIL-88A had a maximal activity of 1554 U/gprotein, which was nearly 5 times higher than free OpdA. Compared with free OpdA, OpdA@MIL-88A exhibited improved organic solvent tolerance, SDS tolerance, thermal stability, and storage stability. OpdA@MIL-88A was used to degrade organophosphorus pesticides on grapes and cucumbers. After reuse 6 times, OpdA@MIL-88A retained more than 66% and 61% of the initial activity, respectively. Therefore, this proposed strategy provided a facile and effective method for degradation of organophosphorus pesticides.

Identification and Characterization of a New Class of (6-4) Photolyase from Vibrio cholerae.

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

Sequential Electrostatic Assembly of a Polymer Surfactant Corona Increases Activity of the Phosphotriesterase arPTE.

We present a new methodology for the generation of discrete molecularly dispersed enzyme-polymer-surfactant bioconjugates. Significantly, we demonstrate that >3-fold increase in the catalytic efficiency of the diffusion-limited phosphotriesterase arPTE can be achieved through sequential electrostatic addition of cationic and anionic polymer surfactants, respectively. Here, the polymer surfactants assemble on the surface of the enzyme via ion exchange to yield a compact corona. The observed rate enhancement is consistent with a mechanism whereby the polymer-surfactant corona gives rise to a decrease in the dielectric constant in the vicinity of the active site of the enzyme, accelerating the rate-determining product diffusion step. The facile methodology has significant potential for increasing the efficiency of enzymes and could therefore have a substantially positive impact for industrial enzymology.

Substrate-imprinted docking of Agrobacterium tumefaciens uronate dehydrogenase for increased substrate selectivity.

Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) belongs to the short-chain dehydrogenase superfamily, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. It is apparently required for the production of D-glucaric acid. AtuUdh-catalyzed reaction is reversible with dual substrate-specific activity (D-galacturonic acid and D-glucuronic acid) in nature. In our study, 34 mutants were pre-screened from 155 mutants generated from AtuUdh (wild-type) and selected 10 structurally stable mutants with increased substrate selectivity. The specificity, efficiency, and selectivity of these mutants for different substrates and cofactors were predicted from 121 docked models using a substrate-imprinted docking approach. Q14F, S36L, and S75T mutants have shown a high binding affinity to D-glucuronic acid and its substrate intermediates such as D-glucaro-1,4-lactone and D-glucaro-1,5-lactone. These mutants exhibited a low binding affinity to the substrate and cofactor required for D-galactaric acid. D34S, N112E and S165E mutants found to show a high selectivity of D-galacturonic acid and its substrate intermediates for D-galactaric acid production. Ser75, Ser165, and Arg174 are active residues playing an imperative role in the substrate selectivity and also contributed in the conjecture the mechanism of transition state stabilization catalyzed by AtuUdh mutants. The present approach was used to reveal the substrate binding mechanism of AtuUdh mutants for a better understanding of the structural basis for selectivity and function.

Development of technologies applied to the biodegradation of warfare nerve agents: Theoretical evidence for asymmetric homogeneous catalysis.

Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.