Product Distribution of Steady-State and Pulsed Electrochemical Regeneration of 1,4-NADH and Integration with Enzymatic Reaction.

The direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD+) results in various products, complicating the regeneration of the crucial 1,4-NADH cofactor for enzymatic reactions. Previous research primarily focused on steady-state polarization to examine potential impacts on product selectivity. However, this study explores the influence of dynamic conditions on the selectivity of NAD+ reduction products by comparing two dynamic profiles with steady-state conditions. Our findings reveal that the main products, including 1,4-NADH, several dimers, and ADP-ribose, remained consistent across all conditions. A minor by-product, 1,6-NADH, was also identified. The product distribution varied depending on the experimental conditions (steady state vs. dynamic) and the concentration of NAD+, with higher concentrations and overpotentials promoting dimerization. The optimal yield of 1,4-NADH was achieved under steady-state conditions with low overpotential and NAD+ concentrations. While dynamic conditions enhanced the 1,4-NADH yield at shorter reaction times, they also resulted in a significant amount of unidentified products. Furthermore, this study assessed the potential of using pulsed electrochemical regeneration of 1,4-NADH with enoate reductase (XenB) for cyclohexenone reduction.

Discovery and Characterization of a Baeyer-Villiger Monooxygenase Using Sequence Similarity Network Analysis.

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

The all-Wales forensic adolescent consultation and treatment service (FACTS): A 5-year referral cohort study.

BACKGROUND: FACTS is a Wales-wide mental health service for 10-17-year-olds with needs beyond the remit of mainstream child and adolescent mental health services (CAMHS). As a purely consultation-liaison service, it differs from other UK services in the field. AIMS: To describe a complete cohort of referrals to FACTS 2013-2017 with service exit by June 2018. METHODS: Clinical, social and offending data were extracted from FACTS records. RESULTS: 80 young people completed a FACTS episode, averaging nearly a year (309 days; range 13-859 days). Mostly boys (65, 81%) of mean age 15.4 years (range 9-18), two-thirds (n = 53) had three or more referral reasons, one invariably being threatened/actual harm to others; only half were criminal-justice involved. Half (41, 51%) were committing sexually harmful acts. Half were self-harming (41, 51%). All but seven had had at least one adverse childhood experience (ACE), nearly half (35, 44%) four or more. Nevertheless, post-traumatic stress disorder (PTSD) was rarely diagnosed (7, 9%); just over one-quarter (23, 29%) had no diagnosis at all. Correspondence analyses endorsed two distinct Attention deficit hyperactivity disorder groups, distinguished by presence/absence of evidenced brain damage or dysfunction. Suicide-related behaviours clustered with the other diagnoses, flashbacks and psychotic symptoms with no diagnosis. Change in home circumstances during a FACTS episode was slight. CONCLUSIONS: The complexity of presenting problems and service involvement evidences need for FACTS. The extent of persistently harmful sexual behaviours is a novel finding, suggesting need for more expert input for this at other service levels. Rarity of PTSD diagnoses was surprising given the extent of ACEs. This raises concerns that services focus on disorder signs rather than the child's inner life. Given the extent of problems, minimal change may be a positive outcome - especially when remaining in the community. Further development of this service should include explicit case-by-case goals and indicative outcome markers.

Mechanism-Based Design of Efficient PET Hydrolases.

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme-substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.

Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Characterization and engineering of a plastic-degrading aromatic polyesterase.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/ß-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Discovery of a new HIV-1 inhibitor scaffold and synthesis of potential prodrugs of indazoles.

A new oxazole scaffold showing great promise in HIV-1 inhibition has been discovered by cell-based screening of an in-house library and scaffold modification. Follow-up SAR study focusing on the 5-aryl substituent of the oxazole core has identified 4k (EC50=0.42µM, TI=50) as a potent inhibitor. However, the analogues suffered from poor aqueous solubility. To address this issue, we have developed broadly applicable potential prodrugs of indazoles. Among them, N-acyloxymethyl analogue 11b displayed promising results (i.e., increased aqueous solubility and susceptibility to enzymatic hydrolysis). Further studies are warranted to fully evaluate the analogues as the potential prodrugs with improved physiochemical and PK properties.

Inhibition of eEF2-K by thieno[2,3-b]pyridine analogues.

Several series of thieno[2-3-b]pyridine analogues were synthesized and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF2-K). Modifications around several regions of the lead molecules were made, with a ring fusion adjacent to the nitrogen on the thienopyridine core being critical for activity. The most active compound 34 shows an IC(50) of 170 nM against eEF2-K in vitro.

Triterpene based compounds with potent anti-maturation activity against HIV-1.

Efforts towards developing orally bioavailable HIV-1 maturation inhibitors starting from betulinic acid 1 are described. SAR resulted in improved potency, physicochemical properties, and enhanced oral absorption in rats.