Donor Substrate Research Articles

Posttranslational regulation of the GCN5 and PCAF acetyltransferases

General control nonderepressible 5 protein (Gcn5) and its homologs, including p300/CBP-associated factor (PCAF), are lysine acetyltransferases that modify both histone and non-histone proteins using acetyl coenzyme A as a donor substrate. While decades of studies have uncovered a vast network of cellular processes impacted by these acetyltransferases, including gene transcription and metabolism, far less is known about how these enzymes are themselves regulated. In this review, we summarize the type and functions of posttranslational modifications proposed to control Gcn5 in both yeast and human cells. We further outline common themes, open questions, and strategies to guide future work.

Bilirubin oxidase as a single enzymatic oxygen scavenger for the development of reductase-based biosensors in the open air and its application on a nitrite biosensor

The commercialization of amperometric or voltammetric biosensors that operate at potentials lower than −0.2 V vs SHE has been hindered by the need for anoxic working conditions due to the interference of molecular oxygen, whose electrochemical reduction can potentially mask other redox processes and generate reactive oxygen species (ROS). A deoxygenation step must be thus integrated into the analytical process. To this end, several (bio)chemical oxygen scavenging systems have been proposed, such as the bi-enzyme system, glucose oxidase/catalase. Still, a few issues persist owing to enzyme impurities and the formation of oxygen reactive species.Here in, we propose a new mono-enzymatic oxygen scavenging system composed of a multicopper oxidase as a single biocatalytic oxygen reducer. As a model, we used bilirubin oxidase (BOD), which catalyzes the direct reduction of oxygen to water in the presence of an electron donor substrate, without releasing hydrogen peroxide. Both the direct electron transfer and mediated electrochemical approach using different co-substrates were screened for the ability to promote the enzymatic reduction of oxygen. An optimal combination of BOD with sodium ascorbate proved to be quick (5 min) and effective. It was subsequently employed, as a proof-of-concept, in a voltammetric biosensor based on a multiheme cytochrome c nitrite reductase, which performs the reduction of nitrite to ammonia at potentials below −0.3 V vs SHE. The nitrite biosensor performed well under ambient air, with no need for a second enzyme to account for the build-up of oxygen reactive intermediaries.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

Enzymatic synthesis of novel unnatural phenoxodiol glycosides with a glycosyl donor flexible glycosyltransferase MeUGT1

Isoflavonoids are of great interest due to their human health-promoting properties, which have resulted in studies on exploiting these phytochemicals as hotspots in diverse bio -industries. Biocatalytic glycosylation of isoflavonoid aglycones to glycosides has attracted marked interests because it enable the biosynthesis of isoflavonoid glycosides with high selectivity under mild conditions, and also provide an environmentally friendly option for the chemical synthesis. Thus, these inspired us to exploit new flexible and effective glycosyltransferases from microbes for making glycosides attractive compounds that are in high demand in several industries. Most recently, we have reported the functional characterization of a bacterial-origin recombinant glycosyltransferase (MeUGT1). Herein, more detailed kinetic characteristics of this biocatalyst, using a number of glycosyl donor substrates, were examined for further investigation of its biocatalytic applicability, enabling it feasible to biosynthesize new glycosides; phenoxodiol-4′-O-α-glucuronide, phenoxodiol-4′-O-α-(2''-N-acetyl)glucosaminide, phenoxodiol-4′-O-α-galactoside, phenoxodiol-4′-O-α-(2''-N-acetyl)galactosaminide and phenoxodiol-4′-O-α-(2''-deoxy)glucoside. The thorough kinetic analyses revealed that while the recombinant enzyme can utilize, albeit with different substrate preference and catalytic efficiency, a total five different nucleotide sugars as glycosyl donors, exhibiting its promiscuity towards glycosyl donors. This is the first report that a recombinant glycosyltransferase MeUGT1 that can regio-specifically glycosylate C4′-hydroxyl function of semi-synthetic phenoxodiol isoflavene to biosynthesize a series of unnatural phenoxodiol-4′-O-α-glycosides.

Formation of ethyl glucuronide by beta-glucuronidase

Substantial amounts of endogenous and xenobiotic compounds are transformed to ß-D-Glucuronides in the human body. The conjugation reaction, which requires uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid as a cosubstrate is catalyzed by UDP-glucuronosyltransferases (UGTs). Beta-glucuronidase (GUSB), which is widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants, opposes this reaction: ß-D-glucuronides are hydrolyzed to ß-D-glucuronic acid and the corresponding aglycon in a configuration retaining manner. This study investigated GUSB activity in the presence of ethanol. Phenolphthalein-ß-D-glucuronide (PHPH-G), 4-nitrophenol-ß-D-glucuronide (4NP-G), morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide were used as substrates. Enzymatic reactions were carried out at 37 °C in buffer solution fortified with different amounts of ethanol (0–70% by volume) using GUSB from different sources ( E. coli , H. pomatia , bovine and human recombinant). The enzymatic reactions were stopped after 60 minutes and all samples were tested for ß-D-ethyl glucuronide (EtG) by a validated LC/MS/MS assay. In incubations of PHPH-G and 4NP-G, the release of aglyca was additionally measured by colorimetrical methods. It was found that EtG, which is a minor metabolite of ethanol but one of the most important direct biomarkers of alcohol consumption in man today, builts up from all investigated ß-D-glucuronides by GUSB activity in the presence of ethanol. The glucuronyl transfer reaction, which was neither observed in absence of ethanol nor in absence of GUSB, is detectable but minor at ethanol concentrations which are commonly observed in blood and tissues after consumption of alcoholic beverages. At higher ethanol concentrations (> 5% by volume), the transfer reaction is predominant and the majority of glucuronyl from ß-D-glucuronides is transferred to ethanol. Different types of donor substrates yielded different amounts of EtG in the presence of the same GUSB-activity and different types of GUSB yielded different amounts of EtG from the same donor substrate. The detection of EtG in several settings independent from human UGT-metabolism (e.g., EtG post collection synthesis in E.coli contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the observed mechanism. A contribution of GUSB activity to EtG kinetics in man is suspected and should be further investigated since high concentrations of ethanol (oral intake of alcoholic beverages) and high concentrations of ß-D-glucuronides (hepatobiliary secretion) are present in the gut, where the main GUSB activity is localized. Finally, the findings could be of interest in context of EtG hair analysis, since the prerequisites for in vitro formation of EtG in this study may also be found on head. There is an additional biochemical path and another source of EtG which deserves further attention in context of alcohol biomarker applications.

Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-β-d-glucopyranosyl β-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of β-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl β-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-β-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl β-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-β-d-glucopyranosyl β-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be β-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-β-d-glucopyranosyl β-d-glucuronide structure released from bacterial and plant acidic carbohydrates.

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow—a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

Modularity of the hydrophobic core and evolution of functional diversity in fold A glycosyltransferases

Hydrophobic cores are fundamental structural properties of proteins typically associated with protein folding and stability; however, how the hydrophobic core shapes protein evolution and function is poorly understood. Here, we investigated the role of conserved hydrophobic cores in fold-A glycosyltransferases (GT-As), a large superfamily of enzymes that catalyze formation of glycosidic linkages between diverse donor and acceptor substrates through distinct catalytic mechanisms (inverting versus retaining). Using hidden Markov models and protein structural alignments, we identify similarities in the phosphate-binding cassette (PBC) of GT-As and unrelated nucleotide-binding proteins, such as UDP-sugar pyrophosphorylases. We demonstrate that GT-As have diverged from other nucleotide-binding proteins through structural elaboration of the PBC and its unique hydrophobic tethering to the F-helix, which harbors the catalytic base (xED-Asp). While the hydrophobic tethering is conserved across diverse GT-A fold enzymes, some families, such as B3GNT2, display variations in tethering interactions and core packing. We evaluated the structural and functional impact of these core variations through experimental mutational analysis and molecular dynamics simulations and find that some of the core mutations (T336I in B3GNT2) increase catalytic efficiency by modulating the conformational occupancy of the catalytic base between “D-in” and acceptor-accessible “D-out” conformation. Taken together, our studies support a model of evolution in which the GT-A core evolved progressively through elaboration upon an ancient PBC found in diverse nucleotide-binding proteins, and malleability of this core provided the structural framework for evolving new catalytic and substrate-binding functions in extant GT-A fold enzymes.

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

Programmable micro-transfer-printing for heterogeneous material integration

Micro-transfer printing is an emerging assembly technique to deterministically transfer ink comprising micro-/nano-objects from a donor substrate to a receiver substrate. This technique has generated increasing interest over the last decade due to its unique capability of integrating diverse heterogeneous materials into various structures and layouts. The growing number of applications enabled by micro-transfer printing includes flexible electronics, sensors, photovoltaics, and micro-LED displays. This work presents a micro-transfer printing approach, which relies on thermally induced adhesion modulation of shape memory polymer materials. An individually addressable micro-fabricated resistive heater array is used to locally deliver the heat for transfer pixel actuation. Selectively transferring chips from a closely packed microchip donor array (pitch: 100 µm, size: 50 × 50 µm2) is demonstrated. The transfer head can be dynamically configured to assemble micro-objects in arbitrary patterns, allowing digital manufacturing, object sorting, or in-line assembly correction of defects. These capabilities, together with the simple and robust structure of the transfer head, can enable high process scalability and flexibility for heterogeneous material integration.

A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases

The transglycosylation behavior and capacity of two clan GH-D α-galactosidases, BoGal36A from the gut bacterium Bacteroides ovatus and Aga27A from the guar plant, was investigated and compared. The enzymes were screened for the ability to use para-nitrophenyl-α-galactoside (pNP-Gal), raffinose and locust bean gum (LBG) galactomannan as glycosyl donors with the glycosyl acceptors methanol, propanol, allyl alcohol, propargyl alcohol and glycerol using mass spectrometry. Aga27A was, in general, more stable in the presence of the acceptors. HPLC analysis was developed and used as a second screening method for reactions using raffinose or LBG as a donor substrate with methanol, propanol and glycerol as acceptors. Time-resolved reactions were set up with raffinose and methanol as the donor and acceptor, respectively, in order to develop an insight into the basic transglycosylation properties, including the ratio between the rate of transglycosylation (methyl galactoside synthesis) and rate of hydrolysis. BoGal36A had a somewhat higher ratio (0.99 compared to 0.71 for Aga27A) at early time points but was indicated to be more prone to secondary (product) hydrolysis in prolonged incubations. The methyl galactoside yield was higher when using raffinose (48% for BoGal36A and 38% for Aga27A) compared to LBG (27% for BoGal36A and 30% for Aga27A).

Parametric investigations on laser-induced forward transfer based micro-3D printing of NiTi alloy

ABSTRACT Laser-induced forward transfer (LIFT)-based micro-3D printing is a process in which the pulsed laser beam is used to transfer the material from thin-film deposited substrate (donor substrate) to the target substrate by inducing a high-pressure gas between the thin film and substrate. This study is focused on printing NiTi shape memory alloy using micro-3D printing for the continuous line pattern deposition. NiTi material is coated in the thin film via sputtering process, and the line pattern is deposited by CO2 laser at a wavelength of 10.6 μm for the transfer process. Numerical simulation is performed to analyze the interface temperature between the thin film and sacrificial layer. The optimized laser fluences for 1.5 μm and 3 μm sacrificial layer thicknesses are 770 mJ/cm2 and 2300 mJ/cm2, respectively. The printed pixel size decreases with an increase in the overlap, and the adhesion of pixels (with substrate) increases with an increase in the target substrate temperature. The transferred pixels are characterized using energy dispersive spectroscopy analysis and X-ray diffraction techniques. The study paves a way for the successful micro-3D printing of NiTi for potential microdevice fabrication.

Synthesis and Evaluation of Three Azide-Modified Disaccharide Oxazolines as Enzyme Substrates for Single-Step Fc Glycan-Mediated Antibody-Drug Conjugation.

Antibody-drug conjugates (ADCs) hold great promise for targeted cancer cell killing. Site-specific antibody-drug conjugation is highly desirable for synthesizing homogeneous ADCs with optimal safety profiles and high efficacy. We have recently reported that azide-functionalized disaccharide oxazolines of the Manβ1,4GlcNAc core were an efficient substrate of wild-type endoglycosidase Endo-S2 for Fc glycan remodeling and conjugation. In this paper, we report the synthesis and evaluation of new disaccharide oxazolines as enzyme substrates for examining the scope of the site-specific conjugation. Thus, azide-functionalized disaccharide oxazolines derived from Manβ1,4GlcNAc, Glcβ1,4GlcNAc, and Galβ1,4GlcNAc (LacNAc) were synthesized. Enzymatic evaluation revealed that wild-type Endo-S2 demonstrated highly relaxed substrate specificity and could accommodate all the three types of disaccharide derivatives for transglycosylation to provide site-specific azide-tagged antibodies, which were readily clicked with a payload to generate homogeneous ADCs. Moreover, we also found that Endo-S2 was able to accommodate drug-preloaded minimal disaccharide oxazolines as donor substrates for efficient glycan transfer, enabling a single-step and site-specific antibody-drug conjugation without the need of an antibody click reaction. The ability of Endo-S2 to accommodate simpler and more easily synthesized disaccharide oxazoline derivatives for Fc glycan remodeling further expanded the scope of this bioconjugation method for constructing homogeneous antibody-drug conjugates in a single-step manner. Finally, cell-based assays indicated that the synthetic homogeneous ADCs demonstrated potent targeted cancer cell killing.

High-Resolution Patterning of Organic Emitting-Layer by Using Inkjet Printing and Sublimation Transfer Process.

We implemented ultra-high resolution patterns of 2822 pixels-per-inch (PPI) via an inkjet printing and vacuum drying process grafted onto a sublimation transfer process. Co-solvented ink with a 1:1 ratio of N,N-dimethylformamide (DMF) to ortho-dichlrorobenzene (oDCB) was used, and the inkjet driving waveform was optimized via analysis of Ohnesorge (Oh)—Reynolds (Re) numbers. Inkjet printing conditions on the donor substrate with 2822 PPI microchannels were investigated in detail according to the drop space and line space. Most sublimation transferred patterns have porous surfaces under drying conditions in an air atmosphere. Unlike the spin-coating process, the drying process of inkjet-printed films on the microchannel has a great effect on the sublimation of transferred thin film. Therefore, to control the morphology, we carefully investigated the drying process of the inkjet-printed inks in the microchannel. Using a vacuum drying process to control the morphology of inkjet-printed films, line patterns of 2822 PPI resolution having a root-mean-square (RMS) roughness of 1.331 nm without voids were successfully fabricated.

SLC3A2 N‐glycosylation and alternate evolutionary trajectories for amino acid metabolism

Cancer and other proliferative diseases are driven by cooperative rewiring of growth signaling and metabolism, where posttranslational modifications to proteins play a critical role in maintaining the disease state. Most PTMs require metabolites as donor substrates, providing underappreciated link between metabolism and signaling. Protein N‐glycosylation and Golgi remodeling of N‐glycan on receptor kinases, T cell receptor, glucagon receptor, and glucose transporters provides feedback between signaling and metabolism. In turn, N‐glycosylation requires uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc), generated by the hexosamine biosynthesis pathway (HBP) from glucose, glutamine and acetyl‐CoA, where the flux of these substrates into UDP‐GlcNAc are in competition with glycolysis, glutaminolysis and fatty‐acid turnover. Thus, N‐glycan branching, a pathway of N‐acetylglucosaminyltransferases (MGAT1, 2, 4 and 5, avian 6) in the Golgi, is a sensor of central metabolism.In a search for additional glycoproteins that link metabolism, N‐glycosylation and signaling, we identified SLC3A2 (4F2hc, CD98),an adaptor to amino acid (AA) transporters SLC7A5‐11, ‐13 which stabilizes cell surface residency. The SLC3A2*SLC7A5 exchanger imports essential AA that stimulate mTOR signaling and anabolic metabolism, while SLC3A2*SLC7A11 supports glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N‐glycans revealed stable site‐specific profiles of Golgi remodeling, with the exception of the conserved N365 where branching and poly‐N‐acetyllactosamine content were sensitive to the insertion of lost ancestral sites and to HBP. The N‐glycans are positioned to promote galectin‐mediated clustering with N‐glycosylated Na++/AA symporters and enhance diffusion‐limited flux between exchangers and AA /Na+ symporters. In a successful experiment of Nature, SLC3A2 has been deleted in Neoaves, a clade representing 95% of Ave species that are adapted for flapping flight, the most metabolically intense form of vertebrate locomotion. Neoaves have also acquired a unique N‐glycan branching specificity GnT‐VI (Mgat6, Q9DGD1), which may support an alternate clustering of N‐glycosylated transporters. KEAP1, and four SLC7A family exchangers are also absent, and two others have duplicated, consistent with a shift in metabolic imperatives of Neoaves. The fate of these genes in Primates, Neoaves and Naked mole rat highlights the tension between metabolic rates and oxidative stress that govern lifestyle and longevity.

Defining pseudoenzymes in glycosylation pathways

Glycosylation pathways regulate diverse cellular processes from protein quality control to host‐pathogen interactions and the enzymes that catalyze glycosidic linkages, glycosyltransferases (GTs), are a large super‐family of proteins that display extensive variation in primary sequence and three‐dimensional structure. A large fraction of GTs also display variations in key active site residues, but the functional and evolutionary implications of these variations are not well understood. In this talk, I will describe our ongoing efforts to map and classify GTs and pseudoGTs across the tree of life. A phylogenetic tree relating diverse GT‐A fold enzymes reveals that pseudoGTs evolved multiple independent times during evolution and variations in the catalytic base (xED‐Asp) correlate with the stereochemistry of glycosidic linkages in the acceptor substrate i.e., inverting vs retaining. Co‐evolutionary analysis reveals that active site variations in GTs are accommodated through compensatory interactions in distal functional sites associated with substrate recognition. Our studies support the “Red Queen” hypothesis in which the selective pressure to catalyze glycosidic linkages between diverse donor and acceptor substrates led to the emergence and expansion of pseudoGTs across the tree of life. Our findings have important implications for understanding other pseudoenzyme families such as pseudokinases.

CDP-ribitol prodrug treatment ameliorates ISPD-deficient muscular dystrophy mouse model

Ribitol-phosphate modification is crucial for the functional maturation of α-dystroglycan. Its dysfunction is associated with muscular dystrophy, cardiomyopathy, and central nervous system abnormalities; however, no effective treatments are currently available for diseases caused by ribitol-phosphate defects. In this study, we demonstrate that prodrug treatments can ameliorate muscular dystrophy caused by defects in isoprenoid synthase domain containing (ISPD), which encodes an enzyme that synthesizes CDP-ribitol, a donor substrate for ribitol-phosphate modification. We generated skeletal muscle-selective Ispd conditional knockout mice, leading to a pathogenic reduction in CDP-ribitol levels, abnormal glycosylation of α-dystroglycan, and severe muscular dystrophy. Adeno-associated virus-mediated gene replacement experiments suggested that the recovery of CDP-ribitol levels rescues the ISPD-deficient pathology. As a prodrug treatment strategy, we developed a series of membrane-permeable CDP-ribitol derivatives, among which tetraacetylated CDP-ribitol ameliorated the dystrophic pathology. In addition, the prodrug successfully rescued abnormal α-dystroglycan glycosylation in patient fibroblasts. Consequently, our findings provide proof-of-concept for supplementation therapy with CDP-ribitol and could accelerate the development of therapeutic agents for muscular dystrophy and other diseases caused by glycosylation defects.

Contact Printing of Multilayered Thin Films with Shape Memory Polymers.

This study describes a method for transfer printing microarrays of multilayered organic–inorganic thin films using shape memory printing stamps and microstructured donor substrates. By applying the films on the microstructured donor substrates during physical vapor deposition and modulating the interfacial adhesion using a shape memory elastomer during printing, this method achieves (1) high lateral and feature-edge resolution and (2) high transfer efficiency from the donor to the receiver substrate. For demonstration, polyurethane-acrylate stamps and silicon/silicon oxide donor substrates were used in the large-area transfer printing of organic–inorganic thin-film stacks with micrometer lateral dimensions and sub-200 nm thickness.

Structure and mechanism of a methyltransferase ribozyme

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

Structural Basis for the Prenylation Reaction of Carbazole-Containing Natural Products Catalyzed by Squalene Synthase-Like Enzymes.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole-3,4-quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ-complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs.