Dual Substrate Research Articles

The discovery and enzymatic characterization of a novel AA10 LPMO from Bacillus amyloliquefaciens with dual substrate specificity

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes, which can catalyze the oxidative cleavage of polysaccharide β-1,4 glycosidic bonds to improve the hydrolysis efficiency of the substrate by other glycoside hydrolases. To improve the conversion efficiency of cellulose and chitin, a strain was screened from the soil of Yuelu Mountain in Hunan province, China. The gene sequence of a novel AA10 LPMO (BaLPMO10) was successfully cloned from the genome of the strain and heterologously expressed in E. coli BL21 (DE3). The optimal enzyme activity of BaLPMO10 was observed at pH 6.0 and 70 °C using 2,6-dimethoxyphenol as substrate, and its maximum specific activity was 91.4 U/g. When BaLPMO10 synergized with glycoside hydrolase to degrade microcrystalline cellulose and colloidal chitin, the reducing sugar content increased by 7% and 23%, respectively, compared to glycoside hydrolase alone. Moreover, the results of molecular docking and molecular dynamics simulation showed that the distance between BaLPMO10 and cellohexaose were further than that of BaLPMO10 and chitohexaose, and the number of hydrogen bonds between BaLPMO10 and cellohexaose were lower than that of BaLPMO10 and chitohexaose. Finally, the hot spot residues of BaLPMO10 interacting with chitohexaose/cellohexaose were identified.

DUAL SUBSTRATE MIMO ANTENNA SYSTEM FOR COGNITIVE RADIO AND AUTOMOTIVE APPLICATIONS

A compact, planar, dual reconfigurable, substrate, multi-band, multiple-input multiple-output (MIMO) antenna system is presented. The proposed MIMO antenna is designed to operate over the entire ultra-wide-band (UWB) frequency band supporting cognitive radio (CR) and automotive applications. The dual-element MIMO antenna is integrated with a radiating element and pure ground plane to operate in two different modes. Two modes of operation are used for the MIMO antenna to sweep the frequency over a wide band especially below 1 GHz. The proposed MIMO antenna covers a wide range of frequency bands from 500MHz~10GHz. The elements of the proposed compact multilayer dual substrate MIMO antenna are placed perpendicular to each other to attain high isolation and polarization diversity which makes the antenna system best suited for CR and automotive applications. The antenna system is developed on dual substrate area of dimensions 58×61×1.6 mm3. The proposed antenna achieves multiband operation with a wide bandwidth of 2GHz within the UWB frequency band.

Unraveling pleiotropic effects of rifampicin by using physiologically based pharmacokinetic modeling: Assessing the induction magnitude of P-glycoprotein-cytochrome P450 3A4 dual substrates.

Rifampicin induces both P‐glycoprotein (P‐gp) and cytochrome P450 3A4 (CYP3A4) through regulating common nuclear receptors (e.g., pregnane X receptor). The interplay of P‐gp and CYP3A4 has emerged to be an important factor in clinical drug–drug interactions (DDIs) with P‐gp–CYP3A4 dual substrates and requires qualitative and quantitative understanding. Although physiologically based pharmacokinetic (PBPK) modeling has become a widely accepted approach to assess DDIs and is able to reasonably predict DDIs caused by CYP3A4 induction and P‐gp induction individually, the predictability of PBPK models for the effect of simultaneous P‐gp and CYP3A4 induction on P‐gp‐CYP3A4 dual substrates remains to be systematically evaluated. In this study, we used a PBPK modeling approach for the assessment of DDIs between rifampicin and 12 drugs: three sensitive P‐gp substrates, seven P‐gp–CYP3A4 dual substrates, and two P‐gp–CYP3A4 dual substrates and inhibitors. A 3.5‐fold increase of intestinal P‐gp abundance was incorporated in the PBPK models to account for rifampicin‐mediated P‐gp induction at steady state. The simulation results showed that accounting for P‐gp induction in addition to CYP3A4 induction improved the prediction accuracy of the area under the concentration‐time curve and maximum (peak) plasma drug concentration ratios compared with considering CYP3A4 induction alone. Furthermore, the interplay of relevant drug‐specific parameters and its impact on the magnitude of DDIs were evaluated using sensitivity analysis. The PBPK approach described herein, in conjunction with robust in vitro and clinical data, can help in the prospective assessment of DDIs involving other P‐gp and CYP3A4 dual substrates. The database reported in the present study provides a valuable aid in understanding the combined effect of P‐gp and CYP3A4 induction during drug development.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Assessing the Functional Redundancy between P-gp and BCRP in Controlling the Brain Distribution and Biliary Excretion of Dual Substrates with PET Imaging in Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are co-localized at the blood–brain barrier, where they display functional redundancy to restrict the brain distribution of dual P-gp/BCRP substrate drugs. We used positron emission tomography (PET) with the metabolically stable P-gp/BCRP substrates [11C]tariquidar, [11C]erlotinib, and [11C]elacridar to assess whether a similar functional redundancy as at the BBB exists in the liver, where both transporters mediate the biliary excretion of drugs. Wild-type, Abcb1a/b(−/−), Abcg2(−/−), and Abcb1a/b(−/−)Abcg2(−/−) mice underwent dynamic whole-body PET scans after i.v. injection of either [11C]tariquidar, [11C]erlotinib, or [11C]elacridar. Brain uptake of all three radiotracers was markedly higher in Abcb1a/b(−/−)Abcg2(−/−) mice than in wild-type mice, while only moderately changed in Abcb1a/b(−/−) and Abcg2(−/−) mice. The transfer of radioactivity from liver to excreted bile was significantly lower in Abcb1a/b(−/−)Abcg2(−/−) mice and almost unchanged in Abcb1a/b(−/−) and Abcg2(−/−) mice (with the exception of [11C]erlotinib, for which biliary excretion was also significantly reduced in Abcg2(−/−) mice). Our data provide evidence for redundancy between P-gp and BCRP in controlling both the brain distribution and biliary excretion of dual P-gp/BCRP substrates and highlight the utility of PET as an upcoming tool to assess the effect of transporters on drug disposition at a whole-body level.

Thermodynamic and Kinetic Modeling of Co-utilization of Glucose and Xylose for 2,3-BDO Production by Zymomonas mobilis.

Prior engineering of the ethanologen Zymomonas mobilis has enabled it to metabolize xylose and to produce 2,3-butanediol (2,3-BDO) as a dominant fermentation product. When co-fermenting with xylose, glucose is preferentially utilized, even though xylose metabolism generates ATP more efficiently during 2,3-BDO production on a BDO-mol basis. To gain a deeper understanding of Z. mobilis metabolism, we first estimated the kinetic parameters of the glucose facilitator protein of Z. mobilis by fitting a kinetic uptake model, which shows that the maximum transport capacity of glucose is seven times higher than that of xylose, and glucose is six times more affinitive to the transporter than xylose. With these estimated kinetic parameters, we further compared the thermodynamic driving force and enzyme protein cost of glucose and xylose metabolism. It is found that, although 20% more ATP can be yielded stoichiometrically during xylose utilization, glucose metabolism is thermodynamically more favorable with 6% greater cumulative Gibbs free energy change, more economical with 37% less enzyme cost required at the initial stage and sustains the advantage of the thermodynamic driving force and protein cost through the fermentation process until glucose is exhausted. Glucose-6-phosphate dehydrogenase (g6pdh), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and phosphoglycerate mutase (pgm) are identified as thermodynamic bottlenecks in glucose utilization pathway, as well as two more enzymes of xylose isomerase and ribulose-5-phosphate epimerase in xylose metabolism. Acetolactate synthase is found as potential engineering target for optimized protein cost supporting unit metabolic flux. Pathway analysis was then extended to the core stoichiometric matrix of Z. mobilis metabolism. Growth was simulated by dynamic flux balance analysis and the model was validated showing good agreement with experimental data. Dynamic FBA simulations suggest that a high agitation is preferable to increase 2,3-BDO productivity while a moderate agitation will benefit the 2,3-BDO titer. Taken together, this work provides thermodynamic and kinetic insights of Z. mobilis metabolism on dual substrates, and guidance of bioengineering efforts to increase hydrocarbon fuel production.

Modelling of metallic inclusions on non-homogeneous dielectric substrates

ABSTRACT In this article, modelling is done for Square Split Ring Resonators (SSRR) as a quasi-static equivalent circuit in a non-homogeneous environment. The authors propose a new relationship for the relative permittivity for a metallic inclusion etched on top of a dual substrate in a non-homogeneous medium. The stacking sequence of the substrates plays a vital role in the response of the SSRR. This paper highlights the change brought about by the dielectric materials’ stacking order on resonant frequency and quality factor. The analytical and numerical study of SSRR etched on the dual substrates shows a good correlation of values. A comparative study indicates a better fit of the proposed formula for the effective permittivity over the conventional averaging method. This article highlights the advantages of using a dual substrate material for fabricating metamaterial structures for different applications. The advantage of the proposed permittivity equation is its applicability to all kinds of geometries. Three different substrate materials were used for the performance study of the SSRR (FR4, Rogers RT/Duroid 5880 and Alumina).

Effects of chronic voluntary alcohol consumption on PDE10A availability: a longitudinal behavioral and [18F]JNJ42259152 PET study in rats.

Phosphodiesterase 10A (PDE10A) is a dual substrate enzyme highly enriched in dopamine-receptive striatal medium spiny neurons, which are involved in psychiatric disorders such as alcohol use disorders (AUD). Although preclinical studies suggest a correlation of PDE10A mRNA expression in neuronal and behavioral responses to alcohol intake, little is known about the effects of alcohol exposure on in vivo PDE10A activity in relation to apparent risk factors for AUD such as decision-making and anxiety. We performed a longitudinal [18F]JNJ42259152 microPET study to evaluate PDE10A changes over a 9-week intermittent access to alcohol model, including 6weeks of alcohol exposure, 2weeks of abstinence followed by 1week relapse. Parametric PDE10A-binding potential (BPND) images were generated using a Logan reference tissue model with cerebellum as reference region and were analyzed using both a volume-of-interest and voxel-based approach. Moreover, individual decision-making and anxiety levels were assessed with the rat Iowa Gambling Task and open-field test over the IAE model. We observed an increased alcohol preference especially in those animals that exhibited poor initial decision-making. The first 2weeks of alcohol exposure resulted in an increased striatal PDE10A binding (> 10%). Comparing PDE10A-binding potential after 2 versus 4weeks of exposure showed a significant decreased PDE10A in the caudate-putamen and nucleus accumbens (pFWE-corrected < 0.05). This striatal PDE10A decrease was related to alcohol consumption and preference. Normalization of striatal PDE10A to initial levels was observed after 1week of relapse, apart from the globus pallidus. This study shows that chronic voluntary alcohol consumption induces a reversible increased PDE10A enzymatic availability in the striatum, which is related to the amount of alcohol preference. Thus, PDE10A-mediated signaling plays an important role in modulating the reinforcing effects of alcohol, and the data suggest that PDE10A inhibition may have beneficial behavioral effects on alcohol intake.

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans-4-hydroxy-L-proline.

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD+ -dependent oxidations of L-glutamate-γ-semialdehyde to L-glutamate (proline metabolism), and 4-hydroxy-L-glutamate-γ-semialdehyde to 4-erythro-hydroxy-L-glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4-hydroxyproline using steady-state kinetics and X-ray crystallography. Trans-4-hydroxy-L-proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L-proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L-stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. The structures suggest that the 10-fold greater preference for the L-stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans-4-hydroxy-L-proline. In contrast, the amine group of the D-stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans-4-hydroxy-L-proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.

A multiband patch antenna with embedded square split ring resonators in non-Homogeneous substrate

The authors have attempted to influence an embedded square split ring resonator (SSRR) response in a stacked non-homogeneous substrate to demonstrate a quad-band antenna. The purpose is to produce multiband operations of a microstrip patch antenna. The highlighted factor is the effect of embedding an SSRR and the differing relative permittivity of the substrate on the side length of the SSRR. The analysis shows that a non-homogeneous dual substrate patch produces multiple bands compared to a single substrate patch antenna without any parameter change. A dual substrate antenna fabricated using FR4 and Rogers RT/Duroid 5880 copper clad sheets with a dimension of 85.6x54x0.908 mm3 (0.314λ0x0.198λ0x0.003λ0). The antenna resonates at 1.1, 2.45, 3.65 and 5.25 GHz in the L-, S- and C-bands. It is possible to employ the patch antenna in WLAN (dual-band) and WiMAX applications and suitable for mobile broadcast service at 1.1 GHz. The authors compare the simulated and measured results of a prototype in the article. The maximum measured gain is 5.48 dBi at 1.1 GHz and 4.025 dBi at 3.65 GHz. The measured bandwidth is 60 MHz (1.2%) at 5.25 GHz.

Additive Manufacturing of a Dual Band, Hybrid Substrate, and Dual Polarization Antenna

We describe the additive manufacturing results pertaining to a multi-function antenna aperture. The antenna consists of customized high dielectric and low loss feedstocks as the enabling technology. The 3D printed prototype shows agreement with simulation while providing excellent performance.

Biochemical characterization of two chitinases from Bacillus cereus sensu lato B25 with antifungal activity against Fusarium verticillioides P03.

Bacterial chitinases are a subject of intense scientific research due to their biotechnological applications, particularly their use as biological pesticides against phytopathogenic fungi as a green alternative to avoid the use of synthetic pesticides. Bacillus cereus sensu lato B25 is a rhizospheric bacterium that is a proven antagonist of Fusarium verticillioides, a major fungal pathogen of maize. This bacterium produces two chitinases that degrade the fungal cell wall and inhibit its growth. In this work, we used a heterologous expression system to purify both enzymes to investigate their biochemical traits in terms of Km, Vmax, optimal pH and temperature. ChiA and ChiB work as exochitinases, but ChiB exhibited a dual substrate activity and it is also an endochitinase. In this work, the direct addition of these chitinases inhibited fungal conidial germination and therefore they may play a major role in the antagonism against F. verticillioides.

EXTH-72. CONTINUOUS INFUSION STUDIES REVEAL THE POTENCY OF ELACRIDAR TO ACT AS A PHARMACO-ENHANCER FOR TREATMENT OF INTRACRANIAL DISEASES BY INHIBITING ABCB1 AND ABCG2 AT THE BLOOD-BRAIN BARRIER

Abstract The blood-brain barrier (BBB) is a formidable hurdle to successful pharmacotherapy of intracranial diseases. ABCB1 and ABCG2 are efflux transporters that play an important role in the BBB, keeping substances out of the brain. Elacridar and tariquidar are third-generation ABCB1-inhibitors developed for treatment of multidrug-resistant tumors. Later, they were shown to also inhibit ABCG2. We aim to improve pharmacotherapy of brain cancer by concomitant use of potentially effective drugs with elacridar. This study was undertaken to determine the relationship between the plasma concentration of the inhibitor and the brain-to-plasma (B/P) ratio of (model) substrate drugs in order to assess which type of drug may best qualify taking into account clinically achievable plasma levels of the inhibitor. We used Abcg2;Abcb1a/b double knockout (DKO), Abcb1a/b KO, Abcg2 KO and wild-type mice receiving a cocktail of 9 drugs at a fixed low dose plus a range of doses of inhibitor by 3-h intraperitoneal infusion to achieve steady-state conditions. DKO mice are the reference for complete inhibition, while single KO mice allow interrogation of the other transporter when using dual substrate drugs. Complete inhibition of Abcb1 by elacridar requires plasma levels of about 1000 nM. Inhibition of Abcg2 is more difficult. For erlotinib and palbociclib about 1000 nM of elacridar is sufficient, but other more profound substrate drugs (e.g. vemurafenib and afatinib) do not reach the B/P ratios achieved in DKO mice, even at 4000 nM. The improvement in B/P ratio that can be reached differs per substrate. Compounds like palbociclib benefit markedly from elacridar with B/P ratios rising from 0.25 to 7, whereas others (e.g. erlotinib and dasatinib) increase from about 0.10 to only 0.40. Thus, elacridar is an efficient pharmaco-enhancer of ABCB1 substrates and weaker ABCG2 substrates, but is not able to improve brain delivery of drugs that are profound ABCG2 substrates.

Trifluoromethyl β‐Enamino Diketones as Dual Substrates for the Synthesis of 5‐Benzoyl‐6‐(trifluoromethyl)pyrimidines and their Pyrimidin‐4(3H)‐one Analogues

A regio‐ and chemoselective method for the preparation of 6‐trifluoromethyl‐5‐benzoyl‐2‐methylsulfanyl pyrimidines and their pyrimidin‐4(3H)‐one analogues, from the cyclocondensation reaction of trifluoromethyl β‐enamino diketones with non‐symmetric 2‐methylisothiourea sulfates is reported. These diketones functioned as dual substrates by providing both products with high regioselectivity (only N3‐substituted isomer was observed) and high chemoselectivity. A new feature provided by the starting material proposed herein is the possibility to either maintain or eliminate the trifluoromethyl group, by choosing the solvent.

Dual Substrate Specificity of the Rutinosidase from Aspergillus niger and the Role of Its Substrate Tunnel

Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free –OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).

Impact of Dual Substrate Limitation on Biodenitrification Modeling in Porous Media

In this work, we consider a model of the biodenitrification process taking place in a spatially-distributed bioreactor, and we take into account the limitation of the kinetics by both the carbon source and the oxidized nitrogen. This model concerns a single type of bacteria growing on nitrate, which splits into adherent bacteria or free bacteria in the liquid, taking all interactions into account. The system obtained consists of four diffusion-convection-reaction equations for which we show the existence and uniqueness of a global solution. The system is approximated by a standard finite element method that satisfies an optimal a priori error estimate. We compare the results obtained for three forms of the growth function: single substrate limiting, “multiplicative” form, and “minimum” form. We highlight the limitation of the ‘ single substrate limiting model”, where the dependency of the bacterial growth on the nitrate is neglected, and find that the “minimum” model gives numerical results closer to the experimental results.

Increased Selectivity for Butanol in Clostridium Pasteurianum Fermentations via Butyric Acid Addition or Dual Feedstock Strategy

Volatility of the petroleum market has renewed research into butanol as an alternate fuel. In order to increase the selectivity for butanol during glycerol fermentation with Clostridium pasteurianum, butyric acid can be added to the medium. In this manuscript, different methods of extracellular butyric acid addition are explored, as well as self-generation of butyric acid fermented from sugars in a co-substrate strategy. Molasses was used as an inexpensive sugar substrate, and the optimal molasses to glycerol ratio was found to allow the butyric acid to be taken back up into the cells and increase the productivity of butanol from all carbon sources. When butyric acid is added directly into the media, there was no significant difference between chemically pure butyric acid, or butyric acid rich supernatant from a separate fermentation. When low concentrations of butyric acid (1 or 2 g/L) are added to the initial media, an inhibitory effect is observed, with no influence on the butanol selectivity. However, when added later to the fermentation, over 1 g/L butyric acid is taken into the cells and increased the relative carbon yield from 0.449 to 0.519 mols carbon in product/mols carbon in substrate. An optimized dual substrate fermentation strategy in a pH-controlled reactor resulted in the relative carbon yield rising from 0.439 when grown on solely glycerol, to 0.480 mols C product/mols C substrate with the dual substrate strategy. An additional benefit is the utilization of a novel source of sugars to produce butanol from C. pasteurianum. The addition of butyric acid, regardless of how it is generated, under the proper conditions can allow for increased selectivity for butanol from all substrates.

Mechanistic investigation in Co-biodegradation of phenanthrene and pyrene by Candida tropicalis MTCC 184

Polycyclic aromatic hydrocarbons like phenanthrene and pyrene are found ubiquitously in terrestrial and aquatic ecosystems. This study has attempted to investigate mechanistic features of simultaneous degradation of phenanthrene and pyrene by the yeast Candida tropicalis. Batch experiments comprising various combinations of initial phenanthrene and pyrene concentrations have been analyzed vis-à-vis kinetic model for cell growth that takes into account self- and cross-inhibition of both substrates. The values of interaction parameters of inhibition revealed strong competitive cross-inhibition between two substrates, due to which biomass yield with dual substrates was reduced significantly. Inhibition induced by pyrene on cell growth was higher than phenanthrene. GC-MS analysis of intermediate metabolites revealed two parallel pathways, first triggered by intracellular cytochrome P450 monooxygenase enzyme, and second initiated by dioxygenase enzymes. The former pathway yielded sulfate conjugate and dihydrodiols, while the latter resulted in acids and aldehydes that enters the TCA cycle.

Biochemical Characterization of an Arginine 2,3‐Aminomutase with Dual Substrate Specificity

Summary of main observation and conclusionThe radical S‐adenosylmethionine (SAM) aminomutases represent an important pathway for the biosynthesis of β‐amino acids. In this study, we report biochemical characterization of BlsG involved in blasticidin S biosynthesis as a radical SAM arginine 2,3‐aminomutase. We showed that BlsG acts on both L‐arginine and L‐lysine with comparable catalytic efficiencies. Similar dual substrate specificity was also observed for the lysine 2,3‐aminomutase from Escherichia coli (LAMEC). The catalytic efficiency of LAMEC is similar to that of BlsG, but is significantly lower than that of the enzyme from Clostridium subterminale (LAMCS), which acts only on L‐lysine rather than on L‐arginine. Moreover, we showed that enzymes can be grouped into two major phylogenetic clades, each corresponding to a certain C3 stereochemistry of the β‐amino acid product. Our study expands the radical SAM aminomutase members and provides insights into enzyme evolution, supporting a trade‐off between substrate promiscuity and catalytic efficiency.

Chlamydia trachomatis Oligopeptide Transporter Performs Dual Functions of Oligopeptide Transport and Peptidoglycan Recycling.

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coliopp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.