Natural photoredox catalysts promote light-driven lytic polysaccharide monooxygenase reactions and enzymatic turnover of biomass.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and chitin and are important for biomass conversion in the biosphere as well as in biorefineries. The target polysaccharides of LPMOs naturally occur in copolymeric structures such as plant cell walls and insect cuticles that are rich in phenolic compounds, which contribute rigidity and stiffness to these materials. Since these phenolics may be photoactive and since LPMO action depends on reducing equivalents, we hypothesized that LPMOs may enable light-driven biomass conversion. Here, we show that redox compounds naturally present in shed insect exoskeletons enable harvesting of light energy to drive LPMO reactions and thus biomass conversion. The primary underlying mechanism is that irradiation of exoskeletons with visible light leads to the generation of H2O2, which fuels LPMO peroxygenase reactions. Experiments with a cellulose model substrate show that the impact of light depends on both light and exoskeleton dosage and that light-driven LPMO activity is inhibited by a competing H2O2-consuming enzyme. Degradation experiments with the chitin-rich exoskeletons themselves show that solubilization of chitin by a chitin-active LPMO is promoted by light. The fact that LPMO reactions, and likely reactions catalyzed by other biomass-converting redox enzymes, are fueled by light-driven abiotic reactions in nature provides an enzyme-based explanation for the known impact of visible light on biomass conversion.

Integrated miRNA-mRNA networks underlie attenuation of chronic ß-adrenergic stimulation-induced cardiac remodeling by minocycline.

Adverse cardiac remodeling contributes to heart failure development and progression, partly due to inappropriate sympathetic nervous system activation. Although ß-adrenergic receptor (ß-AR) blockade is a common heart failure therapy, not all patients respond, prompting exploration of alternative treatments. Minocycline, an FDA-approved antibiotic, has pleiotropic properties beyond antimicrobial action. Recent evidence suggests it may alter gene expression via changes in miRNA expression. Thus, we hypothesized that minocycline could prevent adverse cardiac remodeling induced by the ß-AR agonist isoproterenol, involving miRNA-mRNA transcriptome alterations. Male C57BL/6J mice received isoproterenol (30 mg/kg/day sc) or vehicle via osmotic minipump for 21 days, along with daily minocycline (50 mg/kg ip) or sterile saline. Isoproterenol induced cardiac hypertrophy without altering cardiac function, which minocycline prevented. Total mRNA sequencing revealed isoproterenol altering gene networks associated with inflammation and metabolism, with fibrosis activation predicted by integrated miRNA-mRNA sequencing, involving miR-21, miR-30a, miR-34a, miR-92a, and miR-150, among others. Conversely, the cardiac miRNA-mRNA transcriptome predicted fibrosis inhibition in minocycline-treated mice, involving antifibrotic shifts in Atf3 and Itgb6 gene expression associated with miR-194 upregulation. Picrosirius red staining confirmed isoproterenol-induced cardiac fibrosis, prevented by minocycline. These results demonstrate minocycline's therapeutic potential in attenuating adverse cardiac remodeling through miRNA-mRNA-dependent mechanisms, especially in reducing cardiac fibrosis. NEW & NOTEWORTHY We demonstrate that minocycline treatment prevents cardiac hypertrophy and fibrotic remodeling induced by chronic ß-adrenergic stimulation by inducing antifibrotic shifts in the cardiac miRNA-mRNA transcriptome.

The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

Association between Donor Age and Osteogenic Potential of Human Adipose Stem Cells in Bone Tissue Engineering.

Adipose stem cells (ASCs) have multilineage differentiation capacity and hold great potential for regenerative medicine. Compared to bone marrow-derived mesenchymal stem cells (bmMSCs), ASCs are easier to isolate from abundant sources with significantly higher yields. It is generally accepted that bmMSCs show age-related changes in their proliferation and differentiation potentials, whereas this aspect is still controversial in the case of ASCs. In this review, we evaluated the existing data on the effect of donor age on the osteogenic potential of human ASCs. Overall, a poor agreement has been achieved because of inconsistent findings in the previous studies. Finally, we attempted to delineate the possible reasons behind the lack of agreements reported in the literature. ASCs represent a heterogeneous cell population, and the osteogenic potential of ASCs can be influenced by donor-related factors such as age, but also gender, lifestyle, and the underlying health and metabolic state of donors. Furthermore, future studies should consider experimental factors in in vitro conditions, including passaging, cryopreservation, culture conditions, variations in differentiation protocols, and readout methods.

The impact of kidney stone disease on quality of life in high-risk stone formers.

OBJECTIVE: To assess the impact of kidney stone disease (KSD) and its treatment on the health-related quality of life (HRQOL) of high-risk stone formers with hyperparathyroidism, renal tubular acidosis, malabsorptive disease, and medullary sponge kidney. PATIENTS AND METHODS: The Wisconsin Stone Quality of Life questionnaire was used to evaluate HRQOL in 3301 patients with a history of KSD from 16 institutions in North America between 2014 and 2020. Baseline characteristics and medical history were collected from patients, while active KSD was confirmed through radiological imaging. The high-risk group was compared to the remaining patients (control group) using the Wilcoxon rank-sum test. RESULTS: Of 1499 patients with active KSD included in the study, the high-risk group included 120 patients. The high-risk group had significantly lower HRQOL scores compared to the control group (P < 0.01). In the multivariable analyses, medullary sponge kidney disease and renal tubular acidosis were independent predictors of poorer HRQOL, while alkali therapy was an independent predictor of better HRQOL (all P < 0.01). CONCLUSIONS: Among patients with active KSD, high-risk stone formers had impaired HRQOL with medullary sponge kidney disease and renal tubular acidosis being independent predictors of poorer HRQOL. Clinicians should seek to identify these patients earlier as they would benefit from prompt treatment and prevention.

Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section.

Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers. The method can reveal potential differences in fiber properties along the cross-sectional profile of natural or man-made cellulose fibers. In this analytical approach, carbonyl groups are labeled with a carbonyl selective fluorescence label (CCOA), after which thin fiber layers are sequentially dissolved with the solvent system DMAc/LiCl (9% w/v) and analyzed with size exclusion chromatography coupled with light scattering and fluorescence detection. The analysis of these fractions allowed for the recording of the changes in the chemical structure across the layers, resulting in a detailed cross-sectional profile of the different functionalities and molecular weight distributions. The method was optimized and tested in practice with LPMO (lytic polysaccharide monooxygenase)-treated cotton fibers, where it revealed the depth of fiber modification by the enzyme.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2.

BACKGROUND: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins. RESULTS: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35-37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences. CONCLUSION: All experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

Long-term outcomes of SARS-CoV-2 variants and other respiratory infections: evidence from the Virus Watch prospective cohort in England.

This study compared the likelihood of long-term sequelae following infection with SARS-CoV-2 variants, other acute respiratory infections (ARIs) and non-infected individuals. Participants (n=5,630) were drawn from Virus Watch, a prospective community cohort investigating SARS-CoV-2 epidemiology in England. Using logistic regression, we compared predicted probabilities of developing long-term symptoms (>2 months) during different variant dominance periods according to infection status (SARS-CoV-2, other ARI, or no infection), adjusting for confounding by demographic and clinical factors and vaccination status. SARS-CoV-2 infection during early variant periods up to Omicron BA.1 was associated with greater probability of long-term sequalae (adjusted predicted probability (PP) range 0.27, 95% CI = 0.22-0.33 to 0.34, 95% CI = 0.25-0.43) compared with later Omicron sub-variants (PP range 0.11, 95% CI 0.08-0.15 to 0.14, 95% CI 0.10-0.18). While differences between SARS-CoV-2 and other ARIs (PP range 0.08, 95% CI 0.04-0.11 to 0.23, 95% CI 0.18-0.28) varied by period, all post-infection estimates substantially exceeded those for non-infected participants (PP range 0.01, 95% CI 0.00, 0.02 to 0.03, 95% CI 0.01-0.06). Variant was an important predictor of SARS-CoV-2 post-infection sequalae, with recent Omicron sub-variants demonstrating similar probabilities to other contemporaneous ARIs. Further aetiological investigation including between-pathogen comparison is recommended.

Seasonal Muscular Power Changes: Considerations of Concurrent Resistance and Field-Based Training in Professional Rugby League.

ABSTRACT: Redman, KJ, Wade, L, Whitley, R, Connick, MJ, Kelly, VG, and Beckman, EM. Seasonal muscular power changes: considerations of concurrent resistance and field-based training in professional rugby league. J Strength Cond Res XX(X): 000-000, 2024-A rugby league player's ability to develop or maintain lower-body power will be significantly influenced by the resistance and field-based training loads completed. This study aimed to examine the power changes across a rugby league season and to investigate the relationship of concurrent training and training experience on power changes. Eighteen rugby league players participated in this study. Lower-body power was evaluated using a countermovement jump throughout the season. Four measures of external training loads were collected. A Friedman's test was used to assess differences in power throughout different phases of the season. A series of Spearman's rank-order correlations were conducted to assess the relationship between percentage changes in power during the training block, external training loads, and training experience. Countermovement jump peak velocity significantly decreased from the start (p = 0.006) and end of pre-season (p = 0.022) to the late competition. Correlation analysis revealed a large significant association between early competition field-based external loads (relative (rs = -0.571) and total distance (rs = -0.628)) and change in peak power. Very large significant negative relationships were also observed between mid-competition volume load with change in peak force (rs = -0.825) and peak power (rs = -0.736). Training experience was not significantly associated with changes in muscular power variables throughout this study. Coaches should seek to monitor changes in peak power when competition begins and to assist with decision-making for potential adjustments to running volume and intensity during field-based sessions.

The Effects of Low-Load Squat Jump and Maximal Isometric Priming Exercise on Muscular Performance and Perceptual State.

ABSTRACT: Harrison, PW, James, LP, Jenkins, DG, McGuigan, MR, Holmberg, PM, and Kelly, VG. The effects of low-load squat jump and maximal isometric priming exercise on muscular performance and perceptual state. J Strength Cond Res 38(1): 1-9, 2024-The aim of this study was to examine responses at 3 and 27 hours after low-load jump squat (LL) and maximal isometric half-squat (ISO) priming stimuli. Fifteen resistance-trained males performed LL (4 × 3 at 20% 1 repetition maximum [1RM]), ISO (4 × 3 seconds), and control (CON) activities (standardized warm-up) in a randomized and counterbalanced order. Countermovement jump (CMJ) and isometric midthigh pull tests were conducted to assess performance changes after priming and CON activities. No clear changes in CMJ measures were found after priming activities compared with CON. However, small effect size improvements were found after priming stimuli completed on the same day. A 2.9% decrease in concentric phase duration (CI = 0.3-5.9, p = 0.333, Cliff's delta = -0.156) and a 9.1% increase in RSImod (CI = 0.2-12.3, p = 0.151, Cliff's delta = -0.218) occurred at 3 hours after LL compared with CON. Braking phase duration (CI = 0.8-10.6, p = 0.333, Cliff's delta = -0.213) was 2.9% shorter at 3 hours after ISO compared with CON. No clear changes in isometric peak force occurred after priming activities compared with CON. Additionally, questionnaires were completed to assess perceptual state and perceived effectiveness of the priming stimulus to influence performance. An increase in the "effect of activity" was perceived at 3 hours after LL and ISO (p = 0.013-0.044, Cliff's delta = 0.578-0.6) and at 27 hours after ISO (p = 0.99, Cliff's delta = 0.173) compared with CON. An increase in "muscular heaviness" was also reported at 3 hours after ISO compared with CON (p = 0.199, Cliff's delta = 0.320). The collective findings suggest limited benefits over the day after LL and ISO priming stimuli. However, as there was substantial variation in individual responses, the relative nature of priming responses should be considered when prescribing similar strategies in practical environments.

Effects of Repeated Jump Testing and Diurnal Changes on Subsequent Countermovement Jump and Squat Jump Output and Force-Time Characteristics.

ABSTRACT: Harrison, PW, James, LP, Jenkins, DG, Holmberg, PM, and Kelly, VG. Effects of repeated jump testing and diurnal changes on subsequent countermovement jump and squat jump output and force-time characteristics. J Strength Cond Res 38(1): 174-179, 2024-The aim of this brief study was to investigate the effects of repeated jump testing on performance over 2 consecutive days while considering the possibility of diurnal changes. Fourteen male subjects and 14 recreationally active female subjects completed countermovement jump (CMJ) and squat jump (SJ) testing on 5 occasions (baseline [0,800], 5 minutes [0,820], 8 hours [1,600], 24 hours [0,800], and 32 hours [1,600]) over 32 hours. An additional rested baseline test was conducted on a separate day in the afternoon (1,600) to compare jump performance between morning and afternoon baseline values. Excluding small decreases in CMJ height at 24 hours (p = 0.292, Cliff's delta = -0.225) in male subjects and similar decreases in CMJ height at 5 minutes (p = 0.034, Cliff's delta = -0.245) in addition to SJ height:contraction time at 32 hours (p = 0.126, Cliff's delta = 0.153) in female subjects, findings generally showed no changes in jump performance over multiple assessments. Squat jump metrics may have showed small improvements between morning and afternoon baseline values in male subjects (SJ height:contraction time [p = 0.030, Cliff's delta = 0.225]) and female subjects (SJ height [p = 0.013, Cliff's delta = 0.173] and SJ height:contraction time [p = 0.091, Cliff's delta = 0.163)]. As jump performance was largely unaffected by repeated jump testing, the present findings support the use of monitoring practices and research designs that require multiple jump assessments within acute periods (∼32 hours).

Social factors associated with changes in nutrition risk scores measured using SCREEN-8: data from the Canadian Longitudinal Study on Aging.

Purpose: To examine the social network factors associated with changes in nutrition risk scores, measured by SCREEN-8, over three years, in community-dwelling Canadians aged 45 years and older, using data from the Canadian Longitudinal Study on Aging (CLSA).Methods: Change in SCREEN-8 scores between the baseline and first follow-up waves of the CLSA was calculated by subtracting SCREEN-8 scores at follow-up from baseline scores. Multivariable linear regression was used to examine the factors associated with change in SCREEN-8 score.Results: The mean SCREEN-8 score at baseline was 38.7 (SD = 6.4), and the mean SCREEN-8 score at follow-up was 37.9 (SD = 6.6). The mean change in SCREEN-8 score was -0.90 (SD = 5.99). Higher levels of social participation (participation in community activities) were associated with increases in SCREEN-8 scores between baseline and follow-up, three years later.Conclusions: Dietitians should be aware that individuals with low levels of social participation may be at risk for having their nutritional status decrease over time and consideration should be given to screening them proactively for nutrition risk. Dietitians can develop and support programs aimed at combining food with social participation.

Recognition and evaluation of mental workload in different stages of perceptual and cognitive information processing using a multimodal approach.

This study explores the effects of different perceptual and cognitive information processing stages on mental workload by assessing multimodal indicators of mental workload such as the NASA-TLX, task performance, ERPs and eye movements. Repeated measures ANOVA of the data showed that among ERP indicators, P1, N1 and N2 amplitudes were sensitive to perceptual load (P-load), P3 amplitude was sensitive to P-load only in the prefrontal region during high cognitive load (C-load) states, and P3 amplitude in the occipital and parietal regions was sensitive to C-load. Among the eye movement indicators, blink frequency was sensitive to P-load in all C-load states, but to C-load in only low P-load states; pupil diameter and blink duration were sensitive to both P-load and C-load. Based on the above indicators, the k-nearest neighbours (KNN) algorithm was used to propose a classification method for the four different mental workload states with an accuracy of 97.89%.Practitioner summary: Based on the results of this study, it is possible to implement the monitoring of mental workload states and optimise brain task allocation in operations involving high mental workload, such as human-computer interaction.

Interactive effects of users' openness and robot reliability on trust: evidence from psychological intentions, task performance, visual behaviours, and cerebral activations.

Although trust plays a vital role in human-robot interaction, there is currently a dearth of literature examining the effect of users' openness personality on trust in actual interaction. This study aims to investigate the interaction effects of users' openness and robot reliability on trust. We designed a voice-based walking task and collected subjective trust ratings, task metrics, eye-tracking data, and fNIRS signals from users with different openness to unravel the psychological intentions, task performance, visual behaviours, and cerebral activations underlying trust. The results showed significant interaction effects. Users with low openness exhibited lower subjective trust, more fixations, and higher activation of rTPJ in the highly reliable condition than those with high openness. The results suggested that users with low openness might be more cautious and suspicious about the highly reliable robot and allocate more visual attention and neural processing to monitor and infer robot status than users with high openness.

The study could deepen practitioners' understanding of the effect of openness on trust in robots by examining the psychological intention, task performance, visual behaviours, and physiological activations. Moreover, the interaction effect could provide guidelines for designing robots adaptive to users' personalities, and the multimodal method would be practical for measuring trust in interaction.

Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes.

Selection of the translation initiation site (TIS) is a crucial step during translation. In the 1980s Marylin Kozak performed key studies on vertebrate mRNAs to characterize the optimal TIS consensus sequence, the Kozak motif. Within this motif, conservation of nucleotides in crucial positions, namely a purine at -3 and a G at +4 (where the A of the AUG is numbered +1), is essential for TIS recognition. Ever since its characterization the Kozak motif has been regarded as the optimal sequence to initiate translation in all eukaryotes. We revisit here published in silico data on TIS consensus sequences, as well as experimental studies from diverse eukaryotic lineages, and propose that, while the -3A/G position is universally conserved, the remaining variability of the consensus sequences enables their classification as optimal, strong, and moderate TIS sequences.

Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insights into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage using NMR and circular dichroism spectroscopy. Incubation with ascorbic acid could lead to rearrangements of aromatic residues, followed by more profound structural changes near the copper-active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate.

The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1.

Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity. Mutational effects were assessed by studying EntK1 sensitivity and binding with strains of naturally EntK1-insensitive Lactiplantibacillus plantarum-expressing various RseP variants. We determined that site-directed mutations in conserved sequence motifs related to catalysis and substrate binding, and even deletion of two such motifs known to be involved in substrate binding, did not abolish bacteriocin sensitivity, with one exception. A mutation of a highly conserved asparagine, Asn359, in the extended so-called LDG motif abolished both binding of and killing by EntK1. By constructing various hybrids of the RseP proteins from sensitive E. faecium and insensitive L. plantarum, we showed that the extracellular PDZ domain is the key determinant of EntK1 sensitivity. Taken together, these data may provide valuable insight for guided construction of novel bacteriocins and may contribute to establishing RseP as an antibacterial target.

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

In situ H2 O2 Generation by Choline Oxidase and Its Application in Amino Polysaccharide Degradation by Coupling to Lytic Polysaccharide Monooxygenase.

Chitin, the most abundant amino polysaccharide in Nature, has many applications in different fields. However, processing of this recalcitrant biopolymer in an environmentally friendly manner remains a major challenge. In this context, lytic polysaccharide monooxygenases (LPMOs) are of interest, as they can act on the most recalcitrant parts of chitin and related insoluble biopolymers such as cellulose. Efficient LPMO catalysis can be achieved by feeding reactions with H2 O2 , but careful control of H2 O2 is required to avoid autocatalytic enzyme inactivation. Herein, we present a coupled enzyme system in which a choline oxidase from Arthrobacter globiformis is employed for controlled in situ generation of H2 O2 that fuels LPMO-catalyzed oxidative degradation of chitin. We show that the rate, stability and extent of the LPMO reaction can be manipulated by varying the amount of choline oxidase and/or its substrate, choline chloride, and that efficient peroxygenase reactions may be achieved using sub-µM concentrations of the H2 O2 -generating enzyme. This coupled system requires only sub-stoichiometric amounts of the reductant that is needed to keep the LPMO in its active, reduced state. It is conceivable that this enzyme system may be used for bioprocessing of chitin in choline-based natural deep eutectic solvents.