Poor Substrate Research Articles

Structure-guided discovery of aminopeptidase ERAP1 variants capable of processing antigens with novel PC anchor specificities.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations: S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.

Escherichia coli tRNA (Gm18) methyltransferase (TrmH) requires the correct localization of its methylation site (G18) in the D-loop for efficient methylation.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

Detection of oxaliplatin- and cisplatin-DNA lesions requires different global genome repair mechanisms that affect their clinical efficacy.

The therapeutic efficacy of cisplatin and oxaliplatin depends on the balance between the DNA damage induction and the DNA damage response of tumor cells. Based on clinical evidence, oxaliplatin is administered to cisplatin-unresponsive cancers, but the underlying molecular causes for this tumor specificity are not clear. Hence, stratification of patients based on DNA repair profiling is not sufficiently utilized for treatment selection. Using a combination of genetic, transcriptomics and imaging approaches, we identified factors that promote global genome nucleotide excision repair (GG-NER) of DNA-platinum adducts induced by oxaliplatin, but not by cisplatin. We show that oxaliplatin-DNA lesions are a poor substrate for GG-NER initiating factor XPC and that DDB2 and HMGA2 are required for efficient binding of XPC to oxaliplatin lesions and subsequent GG-NER initiation. Loss of DDB2 and HMGA2 therefore leads to hypersensitivity to oxaliplatin but not to cisplatin. As a result, low DDB2 levels in different colon cancer cells are associated with GG-NER deficiency and oxaliplatin hypersensitivity. Finally, we show that colon cancer patients with low DDB2 levels have a better prognosis after oxaliplatin treatment than patients with high DDB2 expression. We therefore propose that DDB2 is a promising predictive marker of oxaliplatin treatment efficiency in colon cancer.

FlbZIP12 gene enhances drought tolerance via modulating flavonoid biosynthesis in Fagopyrum leptopodum.

Karst lands provide a poor substrate to support plant growth, as they are low in nutrients and water content. Common buckwheat (Fagopyrum esculentum) is becoming a popular crop for its gluten-free grains and their high levels of phenolic compounds, but buckwheat yields are affected by high water requirements during grain filling. Here, we describe a wild population of drought-tolerant Fagopyrum leptopodum and its potential for enhancing drought tolerance in cultivated buckwheat. We determined that the expression of a gene encoding a Basic leucine zipper (bZIP) transcription factor, FlbZIP12, from F. leptopodum is induced by abiotic stresses, including treatment with the phytohormone abscisic acid, salt, and polyethylene glycol. In addition, we show that overexpressing FlbZIP12 in Tartary buckwheat (Fagopyrum tataricum) root hairs promoted drought tolerance by increasing the activities of the enzymes superoxide dismutase and catalase, decreasing malondialdehyde content, and upregulating the expression of stress-related genes. Notably, FlbZIP12 overexpression induced the expression of key genes involved in flavonoid biosynthesis. We also determined that FlbZIP12 interacts with protein kinases from the FlSnRK2 family in vitro and in vivo. Taken together, our results provide a theoretical basis for improving drought tolerance in buckwheat via modulating the expression of FlbZIP12 and flavonoid contents.

Flame retardancy and degradation process of precipitation construction of double-shell flame-retardant microcapsules

In order to address the issues of poor smoke suppression, substrate compatibility, and strong hygroscopicity associated with ammonium polyphosphate, this study employed an in-situ precipitation method to construct a double-shell ammonium polyphosphate microcapsule, with ethyl cellulose and nano-silica serving as shell materials. The microcapsule exhibited improved flame retardancy and fire safety compared to ammonium polyphosphate alone, as evidenced by significant reductions in both maximum heat release rate and total heat release, as determined by thermal-gravimetric analysis (TGA), microcalorimetry (MCC), and vertical burning tests (VBT). Furthermore, the real-time Fourier transform infrared (RT-FTIR), scanning electron microscopy (SEM), and Raman spectrum analyses revealed that the modified kraft paper produced a cross-linked network structure formed by the silica-based substrate and cellulose during combustion, which facilitated the formation of a carbon layer and provided sustained flame retardancy. Physical and hygroscopicity tests demonstrated that the microcapsule exhibited superior compatibility with substrates. Overall, these findings demonstrate that microencapsulation of ammonium polyphosphate effectively addresses the challenges of compatibility and hygroscopicity, while enhancing its flame-retardant efficacy, thus significantly advancing its application in flame retardant materials.

Evaluating the potential of Bacillus licheniformis YZCUO202005 isolated from lichens in maize growth promotion and biocontrol

Lichens exist in an organismal organization of mycobiont, photobiont, and non-photoautotrophic bacteria. These organisms contribute to the growth of lichens even in poor nutrition substrates. However, studies on the isolation and application of non-photoautotrophic bacteria in plant growth and biocontrol are scanty. Therefore, a study was conducted to isolate and evaluate the potential of non-photoautotrophic bacteria from lichen tissues in maize plant growth promotion and biocontrol of plant pathogens (fungi and bacteria). Five bacterial strains were isolated and tested for their ability to produce indole-3-Acetic Acid (IAA). One bacterium named YZCUO202005 produced IAA, siderophores and biofilms, solubilized phosphate and potassium and exhibited extracellular enzymes (cellulases, proteases, amylase, and β −1,3-Glucanase). Based on the 16S rRNA sequence analysis results, YZCUO202005 was identified as Bacillus licheniformis. The strain inhibited the growth of five pathogenic fungi with an inhibition percent of between 58.7% and 71.7% and two pathogenic bacteria. Under greenhouse conditions, YZCUO202005 was tested for its abilities to enhance maize seed germination, and vegetative growth. Compared with the control treatment, the strain significantly enhanced the growth of stem length (i.e. 18 ± 0.64 cm, 78 ± 0.92 cm), leaf length (i.e. 10 ± 0.36 cm, 57 ± 1.42 cm), leaf chlorophyll levels (i.e., 13 ± 0.40, 40 ± 0.43 SPAD), and root length (i.e, 9.8 ± 2.25 cm, 22.5 ± 6.59 cm). Our results demonstrated that B. licheniformis YZCUO202005 from lichens has the potential to promote plant growth and reduce fungal and bacterial pathogens’ growth. Furthermore, the results suggest that lichens are naturally rich sources of plant growth promotion and biocontrol agents that would be used in agriculture.

Molecular basis for bacterial N-glycosylation by a soluble HMW1C-like N-glycosyltransferase

Soluble HMW1C-like N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.

Fabrication of a Printed Heater Using a Composite of Silver Nanowires and Neutral PEDOT:PSS

In this work, a low resistance heater based on a composite of silver nanowires (Ag NWs) and neutral poly (3,4-ethylenedioxythiophene) poly(styrene sulphonate) (PEDOT:PSS) is fabricated. Pure Ag NWs have poor substrate adhesion, poor environmental and thermal stability, and high surface roughness. Local hotspots at wire junctions also leads to uneven joule heating. These disadvantages are overcome by blending the NWs with PEDOT:PSS. Specifically, neutral PEDOT:PSS is used in this work as it is less hygroscopic and possess greater environmental stability compared to the conventionally used acidic PEDOT:PSS. The composite is synthesized as a stable printable dispersion and deposited on a flexible polyethylene terephthalate (PET) substrate using an extrusion-based direct write system. Film thickness was optimized to achieve low sheet resistance and good adhesion and it is laminated with polydimethylsiloxane (PDMS) for environmental protection. The heater shows uniform temperature distribution with a short response time and low operating voltage. Temperature distribution modelling was carried out using COMSOL and correlated well with the experimental data. The heater also exhibits good repeatability and cyclic thermal stability. A prototype device with temperature cut-off and feedback was also implemented using an Arduino controller.

Method for the Synthesis of 2H-1,4-Benzoxazin-3-(4H)-ones via Ligand-Free Copper-Catalyzed Cascade Reaction

AbstractAn efficient method for the synthesis of 2H-1,4-benzoxazin-3-(4H)-ones via ligand-free copper-catalyzed cascade reaction of substituted chloroacetamides with 2-halophenols has been developed for the first time. Some of the issues typically encountered during the synthesis of 2H-1,4-benzoxazin-3-(4H)-ones, including adding noble metals and ligands, poor substrate scope, have been addressed using this newly developed ligand-free copper-catalyzed cascade reaction.

Activation/Inhibition of Cholinesterases by Excess Substrate: Interpretation of the Phenomenological b Factor in Steady-State Rate Equation

Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if b > 1 there is substrate activation, if b < 1 there is substrate inhibition. The mechanistic significance of the b factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines b. However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that b depends on the acylation step. The magnitude of b and opposite b values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the Ω and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.

A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs

The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based invitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

Mistranslation of the genetic code by a new family of bacterial transfer RNAs

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

Iron (III)-doped PbO2 and its application as electrocatalyst for decomposition of phthalocyanine dye.

The textile industry contributes significantly to environmental pollution through dyeing and finishing processes that release dyes into wastewater. Even small amounts of dyes can have harmful effects and cause negative impacts. These effluents have carcinogenic, toxic, and teratogenic properties and can take a long time to be naturally degraded through photo/bio-degradation processes. This work investigates degradation of Reactive Blue 21 (RB21) phthalocyanine dye using anodic oxidation process with PbO2 anode doped with iron III (0.1M) (marked as Ti/PbO2-0.1Fe) and compared with pure PbO2. Ti/PbO2 films with and without doping were successfully prepared by electrodeposition technology on Ti substrates. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM/EDS) was used to characterize the electrode morphology. Also, linear scanning voltammetry (LSV) and cyclic voltammetry (CV) tests were conducted to investigate the electrochemical response of these electrodes. The influence of operational variables on the mineralization efficiency was studied as a function of pH, temperature, and current density. Doping Ti/PbO2 with Fe3+ (0.1M) could reduce the particle to a smaller dimension and slightly increase the oxygen evolution potential (OEP). A large anodic peak was found for both electrodes prepared in the CV test, indicating that oxidation of the RB21 dye was easily achieved on the surface of the prepared anodes. No significant effect of initial pH on the mineralization of RB21 was observed. RB21 decolorization was more rapid at room temperature and increases with increasing current density. A possible degradation pathway for the anodic oxidation of RB21 in aqueous solution is proposed based on the identified reaction products. In general, it can be said that from the findings it was observed that the Ti/PbO2 and Ti/PbO2-0.1Fe electrodes show good performance on RB21 degradation. However, it was noted that the Ti/PbO2 electrode tends to deteriorate over time and exhibits poor substrate adhesion, while the Ti/PbO2-0.1Fe electrode displays superior substrate adhesion and stability.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

A rationale for higher ratios of CH4 to CO2 production in warmer anoxic freshwater sediments and soils

A rationale for higher ratios of <scp>CH₄</scp> to <scp>CO₂</scp> production in warmer anoxic freshwater sediments and soils

Bioconversion and P-gp-Mediated Transport of Depot Fluphenazine Prodrugs after Intramuscular Injection

Fluphenazine (FPZ) decanoate, an ester-type prodrug formulated as a long-acting injection (LAI), is used in the treatment of schizophrenia. FPZ enanthate was also developed as an LAI formulation, but is no longer in use clinically because of the short elimination half-life of FPZ, the parent drug, after intramuscular injection. In the present study, the hydrolysis of FPZ prodrugs was evaluated in human plasma and liver to clarify the reason for this difference in elimination half-lives. FPZ prodrugs were hydrolyzed in human plasma and liver microsomes. The rate of hydrolysis of FPZ enanthate in human plasma and liver microsomes was 15-fold and 6-fold, respectively, faster than that of FPZ decanoate. Butyrylcholinesterase (BChE) and human serum albumin (HSA) present in human plasma, and two carboxylesterase (CES) isozymes, hCE1 and hCE2, expressed in ubiquitous organs including liver, were mainly responsible for the hydrolysis of FPZ prodrugs. FPZ prodrugs may not be bioconverted in human skeletal muscle at the injection site because of lack of expression of BChE and CESs in muscle. Interestingly, although FPZ was a poor substrate for human P-glycoprotein, FPZ caproate was a good substrate. In conclusion, it is suggested that the shorter elimination half-life of FPZ following administration of FPZ enanthate compared with FPZ decanoate can be attributed to the more rapid hydrolysis of FPZ enanthate by BChE, HSA and CESs.

Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae.

Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. KEY POINTS: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.

Biochemical Characterization and NMR Study of a PET-Hydrolyzing Cutinase from Fusarium solani pisi.

In recent years, the drawbacks of plastics have become evident, with plastic pollution becoming a major environmental issue. There is an urgent need to find solutions to efficiently manage plastic waste by using novel recycling methods. Biocatalytic recycling of plastics by using enzyme-catalyzed hydrolysis is one such solution that has gained interest, in particular for recycling poly(ethylene terephthalate) (PET). To provide insights into PET hydrolysis by cutinases, we have here characterized the kinetics of a PET-hydrolyzing cutinase from Fusarium solani pisi (FsC) at different pH values, mapped the interaction between FsC and the PET analogue BHET by using NMR spectroscopy, and monitored product release directly and in real time by using time-resolved NMR experiments. We found that primarily aliphatic side chains around the active site participate in the interaction with BHET and that pH conditions and a mutation around the active site (L182A) can be used to tune the relative amounts of degradation products. Moreover, we propose that the low catalytic performance of FsC on PET is caused by poor substrate binding combined with slow MHET hydrolysis. Overall, our results provide insights into obstacles that preclude efficient PET hydrolysis by FsC and suggest future approaches for overcoming these obstacles and generating efficient PET-hydrolyzing enzymes.