Substrate Selectivity Research Articles

Hop2-Mnd1 functions as a DNA sequence fidelity switch in Dmc1-mediated DNA recombination

Homologous recombination during meiosis is critical for chromosome segregation and also gives rise to genetic diversity. Genetic exchange between homologous chromosomes during meiosis is mediated by the recombinase Dmc1, which is capable of recombining DNA sequences with mismatches. The Hop2-Mnd1 complex mediates Dmc1 activity. Here, we reveal a regulatory role for Hop2-Mnd1 in restricting substrate selection. Specifically, Hop2-Mnd1 upregulates Dmc1 activity with DNA substrates that are either fully homologous or contain DNA mismatches, and it also acts against DNA strand exchange between substrates solely harboring microhomology. By isolating and examining salient Hop2-Mnd1 separation-of-function variants, we show that suppressing illegitimate DNA recombination requires the Dmc1 filament interaction attributable to Hop2-Mnd1 but not its DNA binding activity. Our study provides mechanistic insights into how Hop2-Mnd1 helps maintain meiotic recombination fidelity.

Functional characterization and unraveling the structural determinants of novel steroid hydroxylase CYP154C7 from Streptomyces sp. PAMC26508

This study characterized cytochrome P450 enzyme CYP154C7 from Streptomyces sp. PAMC26508, emphasizing its capability to hydroxylate steroids, especially at the 16α-position. The enzymatic assay of CYP154C7 demonstrated effective conversion across a pH range of 7.2–7.6, with optimal activity at 30 °C in the Pdx/PdR plus NADH system. Kinetic analysis on most converted steroids (androstenedione and adrenosterone) was performed which shows a greater affinity for androstenedione (Km, 11.06 ± 1.903 μM; Vmax, 0.0062 ± 0.0002 sec−1) compared to adrenosterone (Km, 34.50 ± 6.2 μM; Vmax, 0.0119 ± 0.0007 sec−1). A whole-cell system in Escherichia coli, overexpressing recombinant CYP154C7, achieved substantial conversion for steroids, indicating that CYP154C7 can also be used as a potential whole-cell biocatalyst. To gain structural insights, homology models of CYP154C7 and its homologs were constructed using CYP154C5 (PDB ID: 6TO2), refined, validated, and used for docking studies. Comparative docking analysis suggests that lysine (K236) in the active site and tyrosine (Y197) in the substrate access channel of CYP154C7 are crucial for substrate selectivity and catalytic efficiency. This study suggests that CYP154C7 could be a promising candidate for developing modified steroids, providing valuable insights for protein engineering to design commercially useful CYP steroid hydroxylases with diverse substrate specificities.

Ancestral Sequence Reconstruction to Enable Biocatalytic Synthesis of Azaphilones.

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry. Thus, an effective strategy for streamlining the process from enzyme identification to implementation is essential to expanding biocatalysis. Here, we present a strategy combining bioinformatics-guided enzyme mining and ancestral sequence reconstruction (ASR) to resurrect enzymes for biocatalytic synthesis. Specifically, we achieve an enantioselective synthesis of azaphilone natural products using two ancestral enzymes: a flavin-dependent monooxygenase (FDMO) for stereodivergent oxidative dearomatization and a substrate-selective acyltransferase (AT) for the acylation of the enzymatically installed hydroxyl group. This cascade, stereocomplementary to established chemoenzymatic routes, expands access to enantiomeric linear tricyclic azaphilones. By leveraging the co-occurrence and coevolution of FDMO and AT in azaphilone biosynthetic pathways, we identified an AT candidate, CazE, and addressed its low solubility and stability through ASR, obtaining a more soluble, stable, promiscuous, and reactive ancestral AT (AncAT). Sequence analysis revealed AncAT as a chimeric composition of its descendants with enhanced reactivity likely due to ancestral promiscuity. Flexible receptor docking and molecular dynamics simulations showed that the most reactive AncAT promotes a reactive geometry between substrates. We anticipate that our bioinformatics-guided, ASR-based approach can be broadly applied in target-oriented synthesis, reducing the time required to develop biocatalytic steps and efficiently access superior biocatalysts.

Material Selection and Design Methods for Flexible RFID Tag Antenna

In recent years, advancements in flexible printed electronics have significantly propelled the development of wearable devices, especially in areas of integration, comfort, and streamlined manufacturing. By incorporating electronic components and conductive coatings onto flexible substrates, circuits gain enhanced flexibility and some degree of stretchability. Among these circuits, radio frequency identification (RFID) tag antennas have emerged as a focal point in wearable device communication due to their adaptability in power selection and their compatibility with printed circuit technologies. When designing flexible and stretchable RFID tag antennas using printed electronics, two critical factors need to be addressed. The first is the antenna’s structural design, which must account for varying environments and usage contexts. The second is the development of the antenna prototype, involving the selection of conductive coatings and flexible substrates, as well as the printing processes and performance evaluation standards. This paper will explore common conductive coatings and substrate materials used in flexible printed electronics, along with the design methods of printed tag antennas for wearable applications. Lastly, a novel approach is proposed in which the flexible and stretchable RFID tag antenna itself functions as a strain sensor for posture or pressure detection. Unlike conventional strain sensors, this design eliminates the need for additional communication modules, offering a simplified structure that can be easily printed onto clothing, thereby streamlining the production process.

BACE1 Inhibitors for Alzheimer's Disease: Current Challenges and Future Perspectives.

Disease-modifying therapies (DMT) for Alzheimer's disease (AD) are highly longed-for. In this quest, anti-amyloid therapies take center stage supported by genetic facts that highlight an imbalance between production and clearance of amyloid-β peptide (Aβ) in AD patients. Indeed, evidence from basic research, human genetic and biomarker studies, suggests the accumulation of Aβ as a driver of AD pathogenesis and progression. The aspartic protease β-site AβPP cleaving enzyme (BACE1) is the initiator for Aβ production. Underpinning a critical role for BACE1 in AD pathophysiology are the elevated BACE1 concentration and activity observed in the brain and body fluids of AD patients. Therefore, BACE1 is a prime drug target for reducing Aβ levels in early AD. Small-molecule BACE1 inhibitors have been extensively developed for the last 20 years. However, clinical trials with these molecules have been discontinued for futility or safety reasons. Most of the observed adverse side effects were due to other aspartic proteases cross-inhibition, including the homologue BACE2, and to mechanism-based toxicity since BACE1 has substrates with important roles for synaptic plasticity and synaptic homeostasis besides amyloid-β protein precursor (AβPP). Despite these setbacks, BACE1 persists as a well-validated therapeutic target for which a specific inhibitor with high substrate selectivity may yet to be found. In this review we provide an overview of the evolution in BACE1 inhibitors design pinpointing the molecules that reached advanced phases of clinical trials and the liabilities that precluded adequate trial effects. Finally, we ponder on the challenges that anti-amyloid therapies must overcome to achieve clinical success.

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography.

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyzes the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.

Breathable and Stretchable Epidermal Electronics for Health Management: Recent Advances and Challenges.

Advanced epidermal electronic devices, capable of real-time monitoring of physical, physiological, and biochemical signals and administering appropriate therapeutics, are revolutionizing personalized healthcare technology. However, conventional portable electronic devices are predominantly constructed from impermeable and rigid materials, which thus leads to the mechanical and biochemical disparities between the devices and human tissues, resulting in skin irritation, tissue damage, compromised signal-to-noise ratio (SNR), and limited operational lifespans. To address these limitations, a new generation of wearable on-skin electronics built on stretchable and porous substrates has emerged. These substrates offer significant advantages including breathability, conformability, biocompatibility, and mechanical robustness, thus providing solutions for the aforementioned challenges. However, given their diverse nature and varying application scenarios, the careful selection and engineering of suitable substrates is paramount when developing high-performance on-skin electronics tailored to specific applications. This comprehensive review begins with an overview of various stretchable porous substrates, specifically focusing on their fundamental design principles, fabrication processes, and practical applications. Subsequently, a concise comparison of various methods is offered to fabricate epidermal electronics by applying these porous substrates. Following these, the latest advancements and applications of these electronics are highlighted. Finally, the current challenges are summarized and potential future directions in this dynamic field are explored.

Enantioselective photochemical reactions within the confined cavities of supramolecular assemblies

Compared to bulk solvents, reactions in the confined spaces of supramolecular self-assemblies feature rate acceleration, high efficiency and substrate selectivity. These advantages lead to efficient catalytic efficiency and excellent selectivity in enantioselective supramolecular photochemical transformations. During the last few years, enantioselective supramolecular photocatalysis has developed into one of the most powerful strategies to construct enantioenriched chiral compounds. In this review, the recent advances of enantioselective photochemical reactions taking place within the confined spaces of supramolecular assemblies are summarized, with an emphasis on the specific catalytic modes and chemical transformations. Organization of the data follows a subdivision according to supramolecular host and reaction type. At last, the current limitations and the future research orientation of this research field are discussed.

Metabolic gatekeepers: Dynamic roles of sugar transporters in insect metabolism and physiology.

Sugars play multiple critical roles in insects, serving as energy sources, carbon skeletons, osmolytes and signalling molecules. The transport of sugars from source to sink via membrane proteins is essential for the uptake, distribution and utilization of sugars across various tissues. Sugar supply and distribution are crucial for insect development, flight, diapause and reproduction. Insect sugar transporters (STs) share significant structural and functional similarities with those in mammals and other higher eukaryotes. However, they exhibit unique characteristics, including differential regulation, substrate selectivity and kinetics. Here, we have discussed structural diversity, evolutionary trends, expression dynamics, mechanisms of action and functional significance of insect STs. The sequence and structural diversity of insect STs, highlighted by the analysis of conserved domains and evolutionary patterns, underpins their functional differentiation and divergence. The review emphasizes the importance of STs in insect metabolism, physiology and stress tolerance. It also discusses how variations in transporter regulation, expression, selectivity and activity contribute to functional differences. Furthermore, we have underlined the potential and necessity of studying these mechanisms and roles to gain a deeper understanding of insect glycobiology. Understanding the regulation and function of sugar transporters is vital for comprehending insect metabolism and physiological potential. This review provides valuable insights into the diverse functionalities of insect STs and their significant roles in metabolism and physiology.

Solid-State High Harmonic Generation in Common Large Bandgap Substrate Materials.

Solid-state high harmonic generation (sHHG) spectroscopy is an emerging ultrafast technique for studying key material properties such as electronic structure at and away from equilibrium. sHHG anisotropy measurements, where sHHG spectra are recorded depending on the driving electric field relative to the crystal lattice, have become a powerful tool for studying crystal symmetries. Previous works on two-dimensional materials and other quantum materials have often used substrate-supported samples, assuming that all sHHG signals originate from the sample due to the relatively large bandgap of the substrate. While this assumption is generally reasonable, we show that some sHHG emissions from commonly used substrates can occur at moderate intensities of the sHHG driving field. In addition, we show that it is essential to consider not only the sHHG yield from a substrate but also its angular dependence relative to the material of interest. Specifically, in this work, the power-dependent and polarization angle-resolved sHHG emissions of fused silica, calcium fluoride, diamond, and sapphire of two different crystalline qualities and orientations are compared using a mid-infrared (MIR) driving field. This empirical characterization aims to guide the substrate selection for sHHG studies of novel materials to minimize the misattribution and interference of substrate-related sHHG emissions, which opens the possibility to study a wider array of materials.

Functional Relationships between L1CAM, LC3, ATG12, and Aβ.

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer's disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aβ and reduces the Aβ plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aβ and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aβ, bound to L1, is carried to the autophagosome leading to Aβ elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aβ elimination. We propose that enhancing L1 functions may contribute to therapy in humans.

The S2 Pocket Governs the Genus-Specific Substrate Selectivity of Coronavirus 3C-Like Protease.

Coronavirus 3C-like protease (CoV 3CLpro) is essential for viral replication, providing an attractive target for monitoring the evolution of CoV and developing anti-CoV drugs. Here, the substrate-binding modes of 3CLpros from four CoV genera are analyzed and found that the S2 pocket in 3CLpro is highly conserved within each genus but differs between genera. Functionally, the S2 pocket, in conjunction with S4 and S1' pockets, governs the genus-specific substrate selectivity of 3CLpro. Resurrected ancestral 3CLpros from four CoV genera validate the genus-specific divergence of S2 pocket. Drawing upon the genus-specific S2 pocket as evolutionary marker, eight newly identified 3CLpros uncover the ancestral state of modern 3CLpro and elucidate the possible evolutionary process for CoV. It is also demonstrated that the S2 pocket is highly correlated with the genus-specific inhibitory potency of PF-07321332 (an FDA-approved drug against COVID-19) on different CoV 3CLpros. This study on 3CLpro provides novel insights to inform evolutionary mechanisms for CoV and develop genera-specific or broad-spectrum drugs against CoVs.

Optimizing greywater treatment in green walls: A comparative analysis of recycled substrate materials

The utilization of green walls for on-site greywater treatment in urban buildings has garnered significant attention in recent research, with substrates playing a vital role in treatment performance. The incorporation of recycled materials as substrates not only offers a cost-effective solution for greywater treatment, but also enhances sustainability by repurposing wastes. This study investigates the efficacy of three recycled substrates: alum sludge, garden compost, and a mixture of perlite-coconut fiber in three green wall systems for greywater treatment. Each substrate was allocated to a three-pot column. Comparative analysis revealed alum sludge (C3) and the coconut fiber-perlite mix (C1) achieving the highest chemical oxygen demand (COD) removal at 89.45 ± 2.92 % and 89.34 ± 2.47 %, respectively, with garden compost (C2) recording 67.98 ± 7.45 %. For total nitrogen (TN) removal, C2 achieved the lowest efficiency (49.27 ± 9.39 %), while C1 demonstrated 83.79 ± 10.45 %. Notably, C3 exhibited superior total phosphorus (TP) removal at 94.86 ± 1.87 %, compared to 52.07 ± 17.88 % for C1 and 24.90 ± 30.95 % for C2. Our findings demonstrate that substrate selection is crucial for optimizing both pollutant removal and plant health in green walls. The coconut fiber mix emerged as the most effective substrate, supporting robust plant growth and superior pollutant removal, particularly for nitrogen removal. In contrast, garden compost proved unsuitable, with minimal plant survival post-greywater introduction. Alum sludge showed significant promise, particularly for TP removal, reinforcing its effectiveness in green walls. These insights underscore the importance of substrate optimization in greywater treatment systems in urban environments.

9179 Type 2 Diabetes Alters Metabolic Fuel Selection During Intermittent Fasting

Abstract Disclosure: M.O. Conn: None. D.M. Marko: None. J.D. Schertzer: None. Type 2 Diabetes Alters Metabolic Fuel Selection During Intermittent Fasting Diabetes is characterized by elevated blood glucose resulting from defective insulin secretion and/or insulin resistance. Intermittent fasting, a popular weight loss strategy involving periodic reduction of caloric intake, can improve glycemia. Fasting shifts metabolism from carbohydrates to free fatty acids and ketones. However, high blood insulin can inhibit lipolysis and alter metabolic substrate selection in prediabetes, resulting in muscle catabolism to maintain energy homeostasis. Clinical studies have observed a significant decrease in muscle mass in subjects with obesity and diabetes. Our research aims to characterize lean mass versus fat loss after intermittent fasting in a mouse model of obesity and type 2 diabetes (T2D) and investigate the role of muscle alanine catabolism. We hypothesized that intermittent fasting would promote less lipolysis and more muscle alanine catabolism, causing more muscle loss in diabetic mice than controls. We compared male db/db mice manifesting obesity, hyperglycemia, and hyperinsulinemia to age-matched db/+ (control) mice. Our lab established a 5:2 repeated fasting protocol of ad-libitum access to food for 5 days a week and 2 days of fasting on non-consecutive days. Blood glucose was monitored weekly in fed and fasted states to assess glycemic control. While intermittent fasting lowered fasted blood glucose in both groups, no significant body weight or metabolic tissue mass changes were observed. Metabolic cage data demonstrated increased reliance on fat-based substrates for energy in control mice that underwent intermittent fasting during feeding and fasting periods. Diabetic mice did not show significant changes in the fasted state, but intermittent fasting led to increased reliance on carbohydrate oxidation during re-feeding periods, indicating impaired metabolic flexibility. After 10 weeks, the mice were sacrificed, and metabolic tissue histology (adipose, liver, skeletal muscle) will be performed. We will also analyze serum markers (alanine, fatty acids, glycerol, glucose, insulin) and the activity of the metabolic enzyme alanine transaminase (ALT) in the muscle and liver. This experiment will improve our understanding of how lipolysis and protein breakdown in muscle regulate lean mass versus fat mass loss in obesity and T2D, leading to more effective tailoring of fasting to specific populations. Presentation: 6/3/2024

Evolution and functional divergence of glycosyltransferase genes shaped the quality and cold tolerance of tea plants.

Plant glycosyltransferases (UGTs) play a key role in plant growth and metabolism. Here, we examined the evolutionary landscape among UGTs in 28 fully sequenced species from early algae to angiosperms. Our findings revealed a distinctive expansion and contraction of UGTs in the G and H groups in tea (Camellia sinensis), respectively. Whole-genome duplication and tandem duplication events jointly drove the massive expansion of UGTs, and the interplay of natural and artificial selection has resulted in marked functional divergence within the G group of the sinensis-type tea population. In Cluster II of group G, differences in substrate selection (e.g., Abscisic Acid) of the enzymes encoded by UGT genes led to their functional diversification, and these genes influence tolerance to abiotic stresses such as low temperature and drought via different modes of positive and negative regulation, respectively. UGTs in Cluster III of the G group have diverse aroma substrate preferences, which contributes a diverse aroma spectrum of the sinensis-type tea population. All Cluster III genes respond to low-temperature stress, whereas UGTs within Cluster III-1, shaped by artificial selection, are unresponsive to drought. This suggests that artificial selection of tea plants focused on improving quality and cold tolerance as primary targets.

The dimer of human SVCT1 is key for transport function

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na+ dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity.

Regulation of human GnT-IV family activity by the lectin domain

N-Glycan branching critically regulates glycoprotein functions and is involved in various diseases. Among the glycosyltransferases involved in N-glycan branching is the human N-acetylglucosaminyltransferase-IV (GnT-IV) family, which has four members: GnT-IVa, GnT-IVb, GnT-IVc, and GnT-IVd. GnT-IVa and GnT-IVb have glycosyltransferase activity that generates the type-2 diabetes-related β1,4-GlcNAc branch on the α1,3-Man arm of N-glycans, whereas GnT-IVc and GnT-IVd do not. Recently, this enzyme family was found to have a unique lectin domain in the C-terminal region, which is essential for enzyme activity toward glycoprotein substrates but not toward free N-glycans. Furthermore, interaction between the lectin domain of GnT-IV and N-glycan attached to GnT-IV enables self-regulation of GnT-IV activity, indicating that the lectin domain plays a unique and pivotal role in the regulation of GnT-IV activity. In this review, we summarize the GnT-IV family's biological functions, selectivity for glycoprotein substrates, and regulation of enzymatic activity, with a focus on its unique C-terminal lectin domain.

Substrate selectivity and inhibition of the human lysyl hydroxylase JMJD7.

Jumonji-C (JmjC) domain-containing protein 7 (JMJD7) is a human Fe(II) and 2-oxoglutarate dependent oxygenase that catalyzes stereospecific C3-hydroxylation of lysyl-residues in developmentally regulated GTP binding proteins 1 and 2 (DRG1/2). We report studies exploring a diverse set of lysine derivatives incorporated into the DRG1 peptides as potential human JMJD7 substrates and inhibitors. The results indicate that human JMJD7 has a relatively narrow substrate scope beyond lysine compared to some other JmjC hydroxylases and lysine-modifying enzymes. The geometrically constrained (E)-dehydrolysine is an efficient alternative to lysine for JMJD7-catalyzed C3-hydroxylation. γ-Thialysine and γ-azalysine undergo C3-hydroxylation, followed by degradation to formylglycine. JMJD7 also catalyzes the S-oxidation of DRG1-derived peptides possessing methionine and homomethionine residues in place of lysine. Inhibition assays show that DRG1 variants possessing cysteine/selenocysteine instead of the lysine residue efficiently inhibit JMJD7 via cross-linking. The overall results inform on the substrate selectivity and inhibition of human JMJD7, which will help enable the rational design of selective small-molecule and peptidomimetic inhibitors of JMJD7.

Enhanced propanethiol biodegradation by an optimized propanethiol oxidoreductase in microbial cells within an electrode bioreactor

Propanethiol oxidoreductase (PTO) is a novel thiol-oxidizing enzyme from Pseudomonas putida S-1 that utilizes thiols as the sole nutrition source. This enzyme has shown application potential in the bioremediation of thiols, its substrate selectivity, however, has not yet been elucidated. This study reveals that PTO exhibits catalytic activity towards ethanethiol, propanethiol, and butanethiol, but not methanethiol or phenylmethanethiol, indicating unique substrate specificity. Through directed evolution and semi-rational design, we engineered a PTO mutant (A54V&G316T) with twice the specific activity towards propanethiol than the wild type (from 17.3 to 40.7μg/mg/h). Applying voltage in electrode bioreactors enhanced the microbial degradation of propanethiol, with the mutant PTO further accelerating this process. Both non-native expression in E. coli and native expression in engineered P. putida S-1 demonstrated the mutant PTO's effectiveness in increasing PT removal rates. The PT degradation efficiency of engineered P. putida S-1 increases by 3-fold compared to the wild-type in the first 5hours. These findings highlight the potential of combining metabolic and electrochemical engineering to enhance bioremediation of toxic compounds. The engineered PTO mutant improves PT degradation efficiency and broadens its application in practical bioremediation strategies.

Structural adaptations for carboxypeptidase activity in putative S9 acylaminoacyl peptidase from Bacillus subtilis

Prolyl oligopeptidase, or S9 (MEROPS) family enzymes are crucial drug targets due to their association with various diseases, neurological disorders, cell growth, and survival. These implications render them an exceptionally fascinating field of research. Despite sharing similar structural features, they exhibit diverse enzyme activities, including endopeptidase, dipeptidyl peptidase, and acylaminoacyl peptidase. Additionally, a few members of the S9 family demonstrate carboxypeptidase activity. A recent study showed that the S9 peptidase of Bacillus subtilis (S9bs) possesses the conserved sequence feature necessary for carboxypeptidase activity despite being annotated as an acylaminoacyl peptidase in the UniProt database. However, the mechanism of action and identity of S9bs as carboxypeptidase remain unclear. Consequently, we focused our studies on thoroughly investigating S9bs for its carboxypeptidase activity. In the present study, we report biochemical and biophysical analyses of S9bs, confirming its identity as a carboxypeptidase. Further, structural analysis reveals the molecular basis of S9bs' carboxypeptidase activity, highlighting the crucial structural elements like the “cavity loop” and the “two-arginine” residues essential for this activity. Additionally, our studies confirmed that S9bs forms a stable tetrameric assembly and established its quaternary molecular arrangement, which reveals the presence of an oligomeric pore. Altogether, these structural features play a are crucial role in substrate selection for S9 carboxypeptidases. Overall, our findings reveal a distinct carboxypeptidase within the S9 family and significantly enhance our understanding of these enzymes. Moreover, this study sheds light on the mechanisms underlying carboxypeptidase activity, offering valuable insights that could contribute to therapeutic and drug design.