Substrate Specificity Research Articles

Broadened substrate specificity of bacterial dipeptidyl-peptidase 7 enables release of half of all dipeptide combinations from peptide N-termini.

Dipeptide production mediated by dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11 plays a crucial role in growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium. Given the particular P1-position specificity of DPPs, it has been speculated that DPP5 or DPP7 might be responsible for degrading refractory P1 amino acids, i.e., neutral (Thr, His, Gly, Ser, Gln) and hydrophilic (Asn) residues. The present results identified DPP7 as an entity that processes these residues, thus ensuring complete production of nutritional dipeptides in the bacterium. Activity enhancement by the P1' residue was observed in DPP7, as well as DPP4 and DPP5. Toward the refractory P1 residues, DPP7 uniquely hydrolyzed HX|LD-MCA (X=His, Gln, or Asn) and their hydrolysis was most significantly suppressed in dpp7 gene-disrupted cells. Additionally, hydrophobic P2 residue significantly enhanced DPP7 activity toward these substrates. The findings propose a comprehensive 20 P1×20 P2 amino acid matrix showing the coordination of four DPPs to achieve complete dipeptide production along with subsidiary peptidases. The present finding of a broad substrate specificity that DPP7 accounts for releasing 48 % (192/400) of N-terminal dipeptides could implicate its potential role in linking periodontopathic disease to related systemic disorders.

Comparative analysis of lipolytic enzymes involved in the surface fermentation of dried katsuobushi by xerophilic molds.

Fermented katsuobushi, a traditional Japanese seasoning, is produced from skipjack tuna through smoking, drying and fermentation by xerophilic Aspergillus molds, primarily Aspergillus chevalieri and Aspergillus pseudoglaucus. In this study, we characterized lipolytic enzymes (cLip_1 to cLip_5 and pLip_1 to pLip_3) to clarify their roles in lipid hydrolysis during katsuobushi production under low water activity. The enzymes showed significant diversity in their activity, stability and substrate specificity, and in the hydrolysis profiles of their reactions with fish oil. Phylogenetic analyses revealed that cLip_5 showed a high identity with pLip_2 (94%) and these enzymes formed a phylogenetic cluster with filamentous fungal lipases. Purified recombinant enzymes (rcLip_1, rcLip_2, rcLip_4 and rcLip_5) and wild-type enzymes (cLip_3 and pLip_3) showed varying substrate preferences toward p-nitrophenyl esters. The addition of glycerol to reduce the water activity in the reaction mixture led to increased activities of rcLip_1 and rcLip_4, but it did not affect the activity of the other three enzymes. Among the tested six enzymes, cLip_5 showed the highest hydrolytic activity toward fish oil. The cLip_5 and pLip_2 gene transcript levels were moderately high in strains MK86 and MK88, respectively. cLip_5 and its homolog pLip_2 were identified as the most promising enzymes for katsuobushi fermentation, because of their high hydrolytic activities toward fish oil and adaptability to low water activity conditions. These findings support the selection of optimal Aspergillus strains as starter cultures to potentially shorten the fermentation time and improve the quality and shelf life of katsuobushi. © 2025 Society of Chemical Industry.

Mutant glycosidases for labeling sialoglycans with high specificity and affinity

Affinity labeling of biomacromolecules is vital for bioimaging and functional studies. However, affinity probes recognizing glycans with high specificity remain scarce. Here we report the development of glycan recombinant affinity binders (GRABs) based on mutant bacterial sialidases, which are enzymatically inactive but preserve stringent specificity for sialoglycan substrates. By mutating a key catalytic residue of Streptococcus pneumoniae neuraminidase A (SpNanA) and Ruminococcus gnavus neuraminidase H (RgNanH), we develop GRAB-Sia and GRAB-Sia3 recognizing total sialoglycans and α2,3-sialosides, respectively. The GRABs exhibit strict substrate and linkage specificity, and tetramerization with streptavidin substantially increases their avidity. The GRABs and tetrameric GRABs (tetra-GRABs) are effective tools for probing sialoglycans in immunoblotting, flow cytometry, immunoprecipitation, and fluorescence imaging. Furthermore, multiplex analysis with tetra-GRABs uncovers spatially distinct sialoglycans in the various mouse organs. This work provides a versatile toolkit for labeling and analyzing sialoglycans with high specificity, sensitivity, and convenience.

One-Pot Hetero-Di-C-Glycosylation of the Natural Polyphenol Phloretin by a Single C-Glycosyltransferase With Broad Sugar Substrate Specificity.

The structural motif of hetero-di-C-glycosyl compound is prominent in plant polyphenol natural products and involves two different glycosyl residues (e.g., β-d-glucosyl, β-d-xylosyl) attached to carbons of the same phenolic ring. Polyphenol hetero-di-C-glycosides attract attention as specialized ingredients of herbal medicines and their tailored synthesis by enzymatic C-glycosylation is promising to overcome limitations of low natural availability and to expand molecular diversity to new-to-nature glycoside structures. However, installing these di-C-glycoside structures with synthetic precision and efficiency is challenging. Here we have characterized the syntheses of C-β-galactosyl-C-β-glucosyl and C-β-glucosyl-C-β-xylosyl structures on the phloroglucinol ring of the natural polyphenol phloretin, using kumquat (Fortunella crassifolia) C-glycosyltransferase (FcCGT). The FcCGT uses uridine 5'-diphosphate (UDP)-galactose (5 mU/mg) and UDP-xylose (0.3 U/mg) at lower activity than UDP-glucose (3 U/mg). The 3'-C-β-glucoside (nothofagin) is ~10-fold less reactive than non-glycosylated phloretin with all UDP-sugars, suggesting the practical order of hetero-di-C-glycosylation as C-galactosylation or C-xylosylation of phloretin followed by C-glucosylation of the resulting mono-C-glycoside. Each C-glycosylation performed in the presence of twofold excess of UDP-sugar proceeds to completion and appears to be effectively irreversible, as evidenced by the absence of glycosyl residue exchange at extended reaction times. Synthesis of C-β-glucosyl-C-β-xylosyl phloretin is shown at 10 mM concentration in quantitative conversion using cascade reaction of FcCGT and UDP-xylose synthase, allowing for in situ formation of UDP-xylose from the more expedient donor substrate UDP-glucuronic acid. The desired di-C-glycoside with Xyl or Gal was obtained as a single product of the synthesis and its structure was confirmed by NMR.

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4.

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Förster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

Comprehensive proteolytic profiling of Aedes aegypti mosquito midgut extracts: Unraveling the blood meal protein digestion system.

To sustain the gonotrophic cycle, the Aedes aegypti mosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquito's reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity of Ae. aegypti blood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of the Ae. aegypti mosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies.

Identification of YBX2 and TSKS As STK33 Interacting Proteins in Testicular Germ Cells.

Spermiogenesis is a unique process, in which round spermatids undergo morphological changes to form spermatozoa. Serine/Threonine Kinase 33 (STK33), a member of the serine/threonine protein kinase family, plays a pivotal role in spermiogenesis, manifested by the infertile phenotype of Stk33 knockout mice and patients carrying STK33 mutations. To date, the mechanism by which STK33 promotes spermiogenesis is not fully understood. Here we aimed to identify germ cell-specific proteins that interact with STK33. Using immunoprecipitation and mass spectrometry, 13 proteins were identified that potentially interact with STK33 in testicular germ cells. By comparing the expression patterns of the candidate genes in testicular germ cells, we selected Y-Box Binding Protein 2 (YBX2) and Testis Specific Serine Kinase Substrate (TSKS) for validation. When co-expressed in cultured cells, TSKS was immunoprecipitated by STK33, and vice versa. Furthermore, STK33 was recruited to the TSKS foci, likely through interaction with TSKS. Although proximity ligation assay demonstrated that STK33 and YBX2 form the complex in germ cells, their interaction was not recapitulated in cultured cells. Phosphorylation assays showed that STK33 was unable to phosphorylate both YBX2 and TSKS in vitro. Overall, these results suggest that STK33 regulates spermiogenesis through TSKS and YBX2, which warrants further investigation in vivo.

Activation is only the beginning: mechanisms that tune kinase substrate specificity.

Kinases are master coordinators of cellular processes, but to appropriately respond to the changing cellular environment, each kinase must recognize its substrates, target only those proteins on the correct amino acids, and in many cases, only phosphorylate a subset of potential substrates at any given time. Therefore, regulation of kinase substrate specificity is paramount to proper cellular function, and multiple mechanisms can be employed to achieve specificity. At the smallest scale, characteristics of the substrate such as its linear peptide motif and three-dimensional structure must be complementary to the substrate binding surface of the kinase. This surface is dynamically shaped by the activation loop and surrounding region of the substrate binding groove, which can adopt multiple conformations, often influenced by post-translational modifications. Domain-scale conformational changes can also occur, such as the interaction with pseudosubstrate domains or other regulatory domains in the kinase. Kinases may multimerize or form complexes with other proteins that influence their structure, function, and/or subcellular localization at different times and in response to different signals. This review will illustrate these mechanisms by examining recent work on four serine/threonine kinases: Aurora B, CaMKII, GSK3β, and CK1δ. We find that these mechanisms are often shared by this diverse set of kinases in diverse cellular contexts, so they may represent common strategies that cells use to regulate cell signaling, and it will be enlightening to continue to learn about the depth and robustness of kinase substrate specificity in additional systems.

Structural basis of SIRT7 nucleosome engagement and substrate specificity

Chromatin-modifying enzymes target distinct residues within histones to finetune gene expression profiles. SIRT7 is an NAD+-dependent deacylase often deregulated in cancer, which deacetylates either H3 lysine 36 (H3K36) or H3K18 with high specificity within nucleosomes. Here, we report structures of nucleosome-bound SIRT7, and uncover the structural basis of its specificity towards H3K36 and K18 deacylation, combining a mechanism-based cross-linking strategy, cryo-EM, and enzymatic and cellular assays. We show that the SIRT7 N-terminus represents a unique, extended nucleosome-binding domain, reaching across the nucleosomal surface to the acidic patch. The catalytic domain binds at the H3-tail exit site, engaging both DNA gyres of the nucleosome. Contacting H3K36 versus H3K18 requires a change in binding pose, and results in structural changes in both SIRT7 and the nucleosome. These structures reveal the basis of lysine specificity, allowing us to engineer SIRT7 towards enhanced H3K18ac selectivity, and provides a basis for small molecule modulator development.

Structural Insights into Broad-Range Polyphosphate Kinase 2-II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis.

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85 % of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49 %. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

Recent Advances in Protein Engineering and Synthetic Applications of Amino Acid Transaminases

Amino acid transaminases (ATs) have garnered considerable attention in recent years as promising biocatalysts for the synthesis of high‐value chiral chemicals, including both natural and non‐canonical amino acids. These enzymes catalyze the transfer of amino groups from amino acids to keto acids, playing a pivotal role in various biological processes and industrial applications. Characterized by their high turnover rates, remarkable enantioselectivity, and broad substrate specificity, ATs exhibit exceptional versatility and potential. This review presents a comprehensive overview of the classification, reaction mechanisms, and activity assays of ATs. More crucially, we delve into the recent advancements in protein engineering of ATs through directed evolution and rational/semi‐rational design strategies, which have been instrumental in addressing limitations such as low catalytic efficiency and stability. Furthermore, we survey the recent synthetic applications of ATs in the production of aliphatic and aromatic amino acids, highlighting smart amino donors and coupling methods that effectively shift the equilibrium of transamination reactions, as well as enzyme cascades that further expand the scope of reactions. By bridging gaps in research on ATs, this review aims to provide valuable insights and guide for future developments in the field of biocatalysis, ultimately fostering their continued utilization and advancement.

RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

Structural Basis for the Catalysis and Substrate Specificity of a LarA Racemase with a Broad Substrate Spectrum

Structural Basis for the Catalysis and Substrate Specificity of a LarA Racemase with a Broad Substrate Spectrum

Characterization of a maltononaose-producing amylopullulanase from Bacillus aryabhattai W310.

The recombinated amylopullulanase of PulW310B, pullulanase from Bacillus aryabhattai W310, was characterized. Sequence analysis of PulW310B showed that PulW310B has type I pullulanase structures including its typical region and the conserved regions of glycoside hydrolase family 13. Moreover, PulW310B was predicted to has typical domains of pullulanase and SSF51445 belonging to tansglycosidase. While the substrate specificity of PulW310B presented that it is not a type I pullulanase. PulW310B could hydrolyze pullulan into maltotriose, maltohexaose, and maltononaose as main products with the proportions of 73.6%, 16.7%, and 6.7%, which indicated that PulW310B has a certain potential on maltooligosaccharides production due to its 4-α-transglycosylation activity that was consistent with its domain analysis. Based on substrate specificity and hydrolytic properties, PulW310B is an amylopullulanase. This is the first report of maltononaose producing activity of amylopullulanase, which will be very useful in food industry. Moreover, PulW310B is a moderate pullulanase with optimal temperature of 40°C and pH of 7.0. 50% of the maximum activity was retained at room temperature (25°C), which indicated that it is an environmentally friendly enzyme in maltooligosaccharides production.

Biochemical and structural insights into pinoresinol hydroxylase from Pseudomonas sp.

The vanillyl alcohol oxidase/p-cresol methylhydroxylase (VAO/PCMH) flavoprotein family comprises a broad spectrum of enzymes capable of catalyzing the oxidative bioconversions of various substrates. Among them, pinoresinol hydroxylase (PinH) from the 4-alkylphenol oxidizing subgroup initiates the oxidative degradation of (+)-pinoresinol, a lignan important for both lignin structure and plant defense. In this study, we present a detailed biochemical and structural characterization of PinH from Pseudomonas sp., with focus on its substrate specificity and product formation. PinH was expressed in E. coli and purified as FAD-containing, soluble protein. The flavoenzyme catalyzes the hydroxylation of both (+)-pinoresinol and eugenol. Structural analysis reveals its dimeric form, non-covalent flavin binding, and a large active site. AlphaFold models of the PinH-cytochrome complex demonstrate cytochrome's dual role in electron transfer and modulating PinH's conformation. A distinctive feature of PinH is a large cavity that hosts its multi-ring (+)-pinoresinol substrate. The capability of converting bulky lignans is particularly attractive for biotechnological applications aimed at producing high-value compounds from phenolic precursors. These insights expand our knowledge on the structure and mechanism of the VAO/PCMH flavoenzyme family members.

Mutational study of a lytic polysaccharide monooxygenase from Myceliophthora thermophila (MtLPMO9F): Structural insights into substrate specificity and regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes for the biotechnological exploitation of lignocellulosic biomass, yet their efficient application depends on the in-depth understanding of their mechanism of action. Here, we describe the structural and mutational characterization of a C4-active LPMO from Myceliophthora thermophila, MtLPMO9F, that belongs to auxiliary activity family 9 (AA9). MtLPMO9F is active on cellulose, cello-oligosaccharides and xyloglucan. The crystal structure of MtLPMO9F catalytic domain, determined at 2.3Å resolution, revealed a double conformation for loop L3 with a potential implication in the formation of aglycon subsites. Product analysis of reactions with cello-oligosaccharides showed a prevalent -4 to +2 binding mode. Subsequent biochemical characterization of 4 MtLPMO9F point mutants further provided insights in LPMO structure-function relationships regarding both substrate binding and the role of second-coordination sphere residues.

Correlation of interface structure and optical properties of Ga(N,As) and Ga(As,Bi) based type-II hetero structures

Correlation of interface structure and optical properties of Ga(N,As) and Ga(As,Bi) based type-II hetero structures

Substrate and inhibitor specificity of Plasmodium nucleoside transporters ENT1 orthologs.

Malaria caused by Plasmodium infection poses a serious hazard to human health. Plasmodium falciparum equilibrative nucleoside transporter 1 (PfENT1), which mediates nucleoside uptake, is essential for the growth and proliferation of Plasmodium parasites, suggesting that PfENT1 is a potential antimalarial target. The promising compound GSK4 effectively inhibits the transport activity of PfENT1, thereby restraining the growth of Plasmodium parasites. However, it still needs to be clarified whether Plasmodium ENT1 orthologs have different selectivities for nucleosides and inhibitors. Here, we systematically compared the nucleoside selectivity of Plasmodium ENT1 orthologs from P.falciparum (PfENT1), Plasmodium berghei (PbENT1), and Plasmodium vivax (PvENT1), revealing that Plasmodium ENT1 orthologs present a distinct nucleoside recognition pattern. In addition, GSK4 robustly binds to PfENT1 and PvENT1 from two human-hosted Plasmodium parasites but has a weakened binding affinity for PbENT1 from mouse-hosted Plasmodium parasites. We further structurally optimized the inhibitor and generated three GSK4 analogs. One of the GSK4 analogs presented a slightly increased binding affinity for PfENT1. This optimization represents a promising advancement for antimalarial drug development, providing a novel foundation for future endeavors in antimalarial drug design.

PTPA localized in the Golgi apparatus plays an important role in osteoblast differentiation.

Regulatory subunits of protein phosphatase 2A (PP2A) define the substrate and functional specificity of the PP2A holoenzyme within specific organelles. While PP2A regulates osteoblast differentiation, the roles and localization of its regulatory subunits in osteoblasts remain unclear. Here, we identified PTPA, a PP2A regulatory protein, predominantly localized to the Golgi apparatus, closely overlapping with the Golgi marker Giantin. Disruption of the Golgi structure by Brefeldin A caused PTPA to disperse into the cytoplasm. PTPA overexpression inhibited osteoblast differentiation by downregulating key transcriptional regulators. The Golgi-specific localization of PTPA suggests it may influence bone-related protein secretion and maintain Golgi integrity. These findings highlight the critical role of PTPA in osteoblast differentiation and its association with the Golgi apparatus.

Surface display and characterization of recombinant α-l-Rhamnosidase from Emiliania huxleyi on Pichia pastoris.

An α-l-Rhamnosidase gene with an open reading frame of 3192bp encoding a 1036-amino acid protein (EhRha) was cloned from Emiliania huxleyi for flavonoid hydrolysis on the cell surface of Pichia pastoris (P. pastoris) strain GS115 by fusing with the anchor protein (AGα1) from Saccharomyces cerevisiae. Fluorescence microscopy and flow cytometry assays revealed that EhRha was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity assay and substrate specificity analysis showed that the enzyme activity of displayed EhRha was 78 U/g (cell wet weight). EhRha demonstrated a preference for the α-1,6 linkage l-rhamnose in hesperidin and rutin as its optimal substrates, while showing low activity towards the α-1,2 linkage l-rhamnose in naringin. Furthermore, EhRha demonstrated optimal activity at pH 7.0 and 30°C, maintaining stability within a pH range of 4.5-9.0 at temperatures below 50°C, and remained functional at temperatures ranging from 15°C-30°C. The enzyme activity was significantly enhanced by the presence of 10mM Mn2+ and Fe3+, whereas 10mM Ca2+ and 1mM Fe3+ had an inhibitory effect. These findings suggested that displayed EhRha holds promise for enhancing the bioavailability of health-beneficial polyphenols in low-temperature processing applications.