Insights into the Functionalization of the Methylsalicyclic Moiety during the Biosynthesis of Chlorothricin by Comparative Kinetic Assays of the Activities of Two KAS III‐like Acyltransferases

Chlorothricin ( CHL ), an archetypal member of the family of spirotetronate antibiotics, possesses a tetronate‐containing pentacyclic aglycone that is conjugated with a modified methylsalicyclic acid ( MSA ) moiety through a disaccharide linkage. MSA is a polyketide product assembled by the iterative type I polyketide synthase ChlB1. Incorporation of this pharmaceutically important moiety into CHL relies on the activities of two distinct β‐Ketoacyl‐ACP synthase III (KAS III)‐like acyltransferases, ChlB3 and ChlB6, which function together to coordinate the transfer of MSA through ChlB2, a discrete acyl carrier protein (ACP). During the maturation of CHL , MSA needs to be further functionalized by C2‐O‐methylation and C5‐chlorination; however, timing of this functionalization process remains poorly understood. In this study, we report comparative kinetic assays of the activities of the two KAS III‐like acyltransferases ChlB3 and ChlB6 using substrates that vary in substitution extent and ACP carrier. ChlB3 prefers to transfer the immediately assembled 6‐methyl‐MSA moiety from ChlB1‐ACP to the discrete ACP ChlB2, from which this moiety is preferred to be transferred directly onto the molecule desmethylsalicyl‐CHL prior to C2‐O‐methylation and C5‐chlorination. Consequently, MSA functionalization appears to occur at the molecule level rather than at the covalently tethered protein level, i.e., ChlB1‐ACP or ChlB2. Both ChlB3 and ChlB6 are flexible in substrate tolerance, holding promise for CHL engineering‐based structural diversity by using variable MSA moiety.     

Catalytic ring opening of acetylcyclopropane by water and alcohols under the action of copper or palladium salts

The possibility of the cleavage of the C—C bond in acetylcyclopropane (ACP) under the action of water or alcohols in the presence of copper or palladium salts was demonstrated for the first time. At 175—180 °C, the reactions proceeded regioselectively with the cleavage of the C(1)—C(2) bond in the cyclopropane ring. The reaction of ACP with water afforded 5-hydroxypentan-2-one, bis(3-acetylpropyl) ether, and furan compounds, whereas the reactions with alcohols proceeded selectively to form 5-alkoxypentan-2-ones. The yields of the latter depend on the nature and structure of the alcohol, the maximum values (98%) being achieved in the case of primary alcohols.     

胆盐与磷酸钙的相互作用

胆汁的pH条件下(pH=6～8)，应该生成无定形磷酸钙(ACP)，而在胆结石中磷酸钙通常以羟基磷灰石的形式出现.利用谱学方法研究了ACP与胆盐的作用.结果表明，胆盐以胶团的形式与ACP作用，在溶液中形成复合胶团，使其溶解度增加.不同类型胆盐与ACP的作用能力不同：脱氧胆酸钠(NaDC) > 牛磺胆酸钠(NaTC) > 胆酸钠(NaC).胆盐与ACP中结合钙的亲和能力大于结合钙的亲和能力，使ACP在胆汁的环境下容易转化为羟基磷灰石.     

Acid Phosphatase Assay with a Wireless Magnetoelastic Biosensor

Abstract

A wireless remote‐query disposable magnetoelastic biosensor is developed for the assay of acid phosphatase (ACP). The sensor was fabricated by applying a layer of pH‐sensitive polymer to a magnetoelastic ribbon and, on top of it, a sensing film containing bovine serum albumin and adenosine‐5′‐monophosphate (5′‐AMP), the substrate of ACP. In response to an externally applied time‐varying magnetic field, the magnetoelastic sensor mechanically vibrates at a characteristic frequency that is inversely dependent on the mass of the attached film. Because the magnetoelastic sensor is magnetostrictive, the mechanical vibrations of the sensor launch magnetic flux that can be detected remotely from the sensor using a pick‐up coil. The ACP‐catalyzed hydrolysis of 5′‐AMP decreases the solution pH, resulting in the polymer shrinking and, consequently, the resonance frequency of the magnetoelastic sensor increasing. The experimental condition was optimized to be 37°C, in 2.5 mM sodium citrate buffer solution (pH 6.5). Using citrate as buffer can enhance the ACP activity, partly offsetting the effect of the buffer capacity on the sensitivity. The kinetic parameter were measured to be: 1.64×10^–3 M (Michaelis constant) and 130 Hz/min (maximum initial rate). This work provides a remote enzymatic assay of ACP. The relative standard deviation in the measurements of six sensors in parallel is 3.4%. The proposed sensor can determine 0.2～1.2 U/ml of ACP.     

Amorphous calcium phosphate/poly(D,L-lactic acid) composite nanofibers: electrospinning preparation and biomineralization

Amorphous calcium phosphate (ACP) has been recognized as an attractive biomaterial due to its bioactivity and biocompatibility. Electrospinning is a simple and low-cost way to fabricate polymer fibers. In this study, ACP nanoparticles with diameters ranging from 20 to 80 nm were synthesized using a simple precipitation method. ACP nanoparticles were hybridized with poly(D,L-lactic acid) (PDLLA) to form ACP/PDLLA composite nanofibers by electrospinning, and different architectures including the nanofibrous mesh and tube consisting of ACP/PDLLA composite nanofibers were obtained and characterized. The biomineralization and cytocompatibility of as-prepared ACP/PDLLA composite nanofibers were evaluated in vitro. Osteoblast-like MG63 cells were seeded on the ACP/PDLLA composite nanofiber meshes to perform the cytocompatibility evaluation. The ACP/PDLLA composite nanofibers exhibited a fast mineralization behavior in the simulated body fluid. The attachment of MG63 cells and cytotoxicity of ACP/PDLLA composite nanofibers were also evaluated, and the experiments indicated good biocompatibility and bioactivity of ACP/PDLLA composite nanofibers.     

Pressure-induced effects in the heterogeneous adsorption of insulin on chromatographic surfaces

The effect of increasing the average column pressure (ACP) on the heterogeneous adsorption of insulin variants on a C18-bonded silica was studied in isocratic reversed-phase HPLC. Adsorption isotherm data of lispro and porcine insulin obtained for values of the ACP ranging from 57 to 237 bar were fitted to the Langmuir-Freundlich and the Tóth equation. The resulting isotherm parameters, including the equilibrium adsorption constant and the heterogeneity index, were next used for the calculation of distribution functions characterizing the energy of interactions between the adsorbed insulin molecules and the stationary phase. It was observed that increasing the pressure by 180 bar causes a broadening of the distribution functions and a shift of the position of their maximum toward lower interaction energies. These findings suggest that, under high pressures, the insulin molecules interact with the stationary phase in a more diversified way than under low pressures. Additionally, the most probable value of the energy of the insulin-surface interactions becomes lower when the ACP increases. The pressure-induced changes in the interaction of insulin variants with the hydrophobic surface are attributed to a possible conformational flexibility of the molecular structure of this protein.     

Amorphous Calcium Phosphate Based Composites: Effect of Surfactants and Poly(ethylene oxide) on Filler and Composite Properties

The uncontrolled aggregation of amorphous calcium phosphate (ACP) particulate fillers and their uneven distribution within polymer matrices can have adverse effects on the properties of ACP composites. In this article, we assessed the influence of nonionic and anionic surfactants and poly(ethylene oxide) (PEO) introduced during the preparation of ACP on the particle size distribution and compositional properties of ACP. In addition, the mechanical strength of polymeric composites utilizing such fillers with a photo‐activated binary methacrylate resin was evaluated. Zirconia‐hybridized ACP (Zr‐ACP) filler and its corresponding composite served as controls for this study. Surfactant‐ and PEO‐ACPs had an average water content of 16.8% by mass. Introduction of the anionic surfactant reduced the median particle diameter about 45% (4.1 µm versus 7.4 µm for the Zr‐ACP control). In the presence of PEO, however, the d_m increased to 14.1 µm. There was no improvement in the biaxial flexure strength (BFS) in any of the dry composite specimens prepared with the surfactant and/or PEO‐ACPs compared to those formulated with Zr‐ACP. The BFS of wet composite specimens decreased by 50% or more after a month‐long exposure to saline solutions. Other types of surfactants and/or polymers as well as alternative surface modification protocols need to be explored for their potential to provide better dispersion of ACP into the matrix resin and better mechanical performance ACP composites.     

Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

In fungal non‐reducing polyketide synthases (NR‐PKS) the acyl‐carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein–protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism‐based crosslinkers for NR‐PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP–PT recognition were identified. We used this information to improve ACP compatibility with non‐cognate PT domains, which resulted in the first gain‐of‐function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.     

Effect of phosvitin on the nucleation and growth of calcium phosphates in physiological solutions

The promoting effect of phosvitin on the nucleation of hydroxyapatite (HAP) and the inhibitory effect of phosvitin on the transformation from amorphous calcium phosphate (ACP) to HAP were investigated. Atomic force microscopy observations showed that the nucleation of HAP on collagen substrate was greatly enhanced when the phosvitin was bound on the collagen surface. Nucleated crystals were uniformly distributed with a high nucleation rate on the collagen surface in the presence of phosvitin, while, in the absence of phosvitin, crystals nucleated slowly and were observed only at some particular area. Time-resolved static light scattering measurements revealed that the transformation from ACP to HAP was inhibited when free phosvitin was present in the calcium phosphate solutions. The transformation kinetics in the absence of phosvitin, which is a direct reconstruction of the inner ACP structure to HAP, was changed to heterogeneous growth of HAP on ACP with time.     

Microwave‐Assisted Hydrothermal Rapid Synthesis of Amorphous Calcium Phosphate Mesoporous Microspheres Using Adenosine 5′‐Diphosphate and Application in pH‐Responsive Drug Delivery

Herein we report a rapid and green strategy for the preparation of amorphous calcium phosphate mesoporous microspheres (ACP‐MSs) using adenosine 5′‐diphosphate disodium salt (ADP) as an organic phosphorus source by a microwave‐assisted hydrothermal method. The effects of the pH value, the reaction time, and temperature on the crystal phase and morphology of the product are investigated. The ADP biomolecules used in this strategy play an important role in the formation of ACP‐MSs. The as‐prepared ACP‐MSs are efficient for anticancer drug delivery by using doxorubicin (Dox) as a model drug, and the Dox‐loaded ACP‐MSs show a high ability to damage cancer cells. Moreover, the ACP‐MSs drug delivery system exhibits a pH‐responsive drug‐release behavior due to the degradation of ACP‐MSs at a low pH value, thus, it is promising for applications in pH‐responsive drug delivery.     

Amorphous Calcium Phosphate Based Composites: Effect of Surfactants and Poly(ethylene oxide) on Filler and Composite Properties

The uncontrolled aggregation of amorphous calcium phosphate (ACP) particulate fillers and their uneven distribution within polymer matrices can have adverse effects on the properties of ACP composites. In this paper we assessed the influence of non-ionic and anionic surfactants and poly(ethylene oxide) (PEO) introduced during the preparation of ACP on the particle size distribution and compositional properties of ACP. In addition, the mechanical strength of polymeric composites utilizing such fillers with a photo-activated binary methacrylate resin was evaluated. Zirconia-hybridized ACP (Zr-ACP) filler and its corresponding composite served as controls for this study. Surfactant- and PEO-ACPs had an average water content of 16.8 % by mass. Introduction of the anionic surfactant reduced the median particle diameter about 45 % (4.1 μm vs. 7.4 μm for the Zr-ACP control). In the presence of PEO, however, the d(m) increased to 14.1 μm. There was no improvement in the biaxial flexure strength (BFS) in any of the dry composite specimens prepared with the surfactant- and/or PEO-ACPs compared to those formulated with Zr-ACP. The BFS of wet composite specimens decreased by 50 % or more after a month-long exposure to saline solutions. Other types of surfactants and/or polymers as well as alternative surface modification protocols need to be explored for their potential to provide better dispersion of ACP into the matrix resin and better mechanical performance ACP composites.     

Minimization of Electrode Adsorption Effects: The Cadmium–Humic Acid System Studied by Phase Sensitive Alternating Current Polarography

The use of phase‐sensitive Alternating Current Polarography (ACP) is investigated for the minimization of adsorption effects in metal‐ligand systems with induced metal adsorption onto the electrode. When ACP is applied to obtain information on metal complexation in bulk, some problems arise from the faradaic contribution of adsorbed species. This effect can be corrected by using the capacitive current measured in the potential region of the faradaic peak. Using this correction, ACP produces similar results to those from Reverse Pulse Polarography (RPP), a technique that minimizes electrode adsorption effects on the measured currents. The method proposed is applied to the study of the Cd–humic acid system that has been investigated by ACP, RPP and Differential Pulse Polarography (DPP).     

The malonyl transferase activity of type II polyketide synthase acyl carrier proteins

Acyl carrier proteins (ACPs) play a fundamental role in directing intermediates among the enzyme active sites of fatty acid and polyketide synthases (PKSs). In this paper, we demonstrate that the Streptomyces coelicolor (S. coelicolor) actinorhodin (act) PKS ACP can catalyze transfer of malonate to type II S. coelicolor fatty acid synthase (FAS) and other PKS ACPs in vitro. The reciprocal transfer from S. coelicolor FAS ACP to a PKS ACP was not observed. Several mutations in both act ACP and S. coelicolor FAS ACP could be classified by their participation in either donation or acceptance of this malonyl group. These mutations indicated that self-malonylation and malonyl transfer could be completely decoupled, implying that they were separate processes and that a FAS ACP could be converted from a non-malonyl-transferring protein to one with malonyl transferase activity.     

[C^N]-Alkenyl Gold(III) Complexes by Proximal Ring-Opening of (2-Pyridyl)alkylidenecyclopropanes: Mechanistic Insights

Pyridine-substituted alkylidenecyclopropanes (Py-ACPs) react with gold(III) salts under mild reaction conditions through an unprecedented, proximal ring-opening pathway, to generate highly appealing, catalytically active pyridine alkenyl [C^N]-gold(III) species. Mechanistic studies reveal that the activation of the C−C bond of the ACP takes place through an unusual concerted, σ-bond metathesis type-process.     

Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis.

BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known. RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process. CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.     

An Unusual Type II Polyketide Synthase System Involved in Cinnamoyl Lipid Biosynthesis

As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier‐protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase‐dependent ACP‐tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP‐tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier‐protein‐dependent biosynthetic machineries.     

Carbon-supported manganese for heterogeneous activation of peroxymonosulfate for the decomposition of phenol in aqueous solutions

Phenolic substances are harmful to humans and other living things, even at low concentrations. Therefore, phenol must be removed from water with the proper process. One of the most effective processes for degrading phenol is heterogeneous catalytic oxidation. Three carbon materials as supports were used to prepare manganese-oxide based catalyst (2.5% MnOx/ACP and 2.5% MnOx/ACN), and graphene oxide (2.5% MnOx/GO). These catalysts were tested for the degradation of phenol in aqueous solution using peroxymonosulfate as a source of sulfate radical. The physio-chemical catalysts were characterised by several characterisation techniques such as powder X-ray diffraction, N₂-sorption (BET), scanning electron microscopy (SEM) equipped with Dispersive Energy X-ray Spectroscopy (EDS). In comparison to other catalysts, heterogeneous activation of peroxymonosulfate was more effectively done by 2.5% MnOx/ACP, resulting in a higher production rate of sulfate radicals. In the presence of a catalyst at 0.2 g and 1 g peroxymonosulfate in 500 mL solution at 25 °C, 90% total organic carbon (TOC) removal and phenol decomposition of 100% was achieved in 90 min with phenol concentration of 75 mg/L. First-order kinetics were followed by phenol decomposition with the energy of activation on 2.5% MnOx/ACP of 15.0 kJ/mol.     

Highly Stable Amorphous Calcium Phosphate Porous Nanospheres: Microwave‐Assisted Rapid Synthesis Using ATP as Phosphorus Source and Stabilizer,and Their Application in Anticancer Drug Delivery

Highly stable amorphous calcium phosphate (ACP) porous nanospheres with a relatively uniform size and an average pore diameter of about 10 nm have been synthesized by using a microwave‐assisted hydrothermal method with adenosine 5′‐triphosphate disodium salt (ATP) as the phosphorus source and stabilizer. The as‐prepared ACP porous nanospheres have a high stability in the phosphate buffer saline (PBS) solution for more than 150 h without phase transformation to hydroxyapatite, and the morphology and size were essentially not changed. The important role of ATP and effects of experimental conditions on the formation of ACP porous nanospheres were also investigated. The ACP porous nanospheres were characterized by X‐ray powder diffraction (XRD), Fourier‐transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). This method is facile, rapid, surfactant‐free and environmentally friendly. The as‐prepared ACP porous nanospheres are efficient for anticancer drug (docetaxel) loading and release. The ACP porous nanosphere drug‐delivery system with docetaxel shows a high ability to damage tumor cells, thus, is promising for the application in anticancer treatment.     

Trapping of a Polyketide Synthase Module after C−C Bond Formation Reveals Transient Acyl Carrier Domain Interactions

Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C−C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C−C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential “parking position” for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C−C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.     

Redispersible and stable amorphous calcium phosphate nanoparticles functionalized by an organic bisphosphate

Although much effort has been focused on the preparation of stable amorphous calcium phosphate （ACP） nanoparticles in aqueous solution, the redispersibility and long-term stability of ACP nanoparticles in aqueous solution remains an unresolved problem. In this work, stable colloidal ACPs were prepared by using an organic bisphosphonate （BP） as a sterically hindered agent in aqueous solution. The harvested calcium phosphate nanoparticles were characterized by inductively coupled plasma atomic emission spectrometry （ICP-AES）, Fourier transform infrared （FTIR）, X-ray diffraction （XRD）, dynamic light scattering （DLS） and transmission electron microscopy （TEM）. ICP-AES, FTIR and XRD results suggested the particles were ACP. DLS and TEM results indicated that the size of the ACP nanoparticles were in the range of 60 nm with a spherical morphology. The resulting calcium phosphate nanoparticles retained its amorphous nature in aqueous solution for at least 6 months at room temperature due to the stabilizing effect of the organic bisphosphonate. Moreover, the surface of the ACP nanoparticles adsorbed with the organic bisphosphate used showed good redispersibility and high colloid stability both in organic and aqueous solutions.