Large Substrates Research Articles

MicroED structure of the C11 cysteine protease clostripain

Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

Electrochemical deposition of coloured Ni/Cu2O bilayer films on large area plastic substrates for decorative applications

ABSTRACT In this study, coloured Ni/Cu2O bilayer films were electrochemically deposited on large area automobile plastic parts (7 × 13 cm) for decorative applications. Such coatings may become an alternative to ubiquitously used Cr coatings for plastics. Purple, dark cyan and gold colours with good homogeneity and high brightness could be produced by tuning the deposition time of the Cu2O layer. Colouration of the bilayer film can be ascribed to interference of light through the metal/dielectric layer, where the thickness of the dielectric layer controls the wavelength of the reflected light. Cu2O film has a compact structure and low roughness, which is essential to obtain uniform colours. Moreover, the Ni/Cu2O bilayer exhibits good adhesion behaviour, environmental stability and enhanced corrosion resistance. This process is industrially viable and enables the large scale production of such decorative coatings.

Shy is a proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy co-purified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When co-expressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to co-purify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retroaldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity towards C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted anti-parallel β-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo)-heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

Enhancing Paenibacillus sp. Cold-Active Acetyl Xylan Esterase Activity through Semi-Rational Protein Engineering

Interest in protein engineering for the enzymatic production of valuable products, such as pharmaceutical compounds and biofuels, is growing rapidly. The cold-active acetyl xylan esterase from Paenibacillus sp. (PbAcE) presents unusually broad substrate specificity. Here, we engineered a hydrophobic substrate-binding pocket to enable the accommodation of relatively large alcohol substrates, such as linalyl acetate and α-terpinyl acetate. To identify candidate residues for engineering, we performed covalent docking of substrates to the Ser185 active site using the HCovDock program. Functional hotspots were analyzed using HotSpot Wizard 3.1. Lys91, His93, and Tyr182 were selected for site-saturation mutagenesis (SSM). After generating the SSM mutant library, a qualitative colorimetric assay was conducted to identify positive mutants. Three, two, and five single mutants were selected for Lys91, His93, and Tyr182, respectively. The best single mutants were then sequentially combined to generate double and triple mutants. Single mutants exhibited a 10–30% increase in activity compared to that of wild-type PbAcE, while no significant synergistic improvements were observed in the double and triple mutants. The increase in activity against both linalyl acetate and α-terpinyl acetate was similar. Mutation did not affect the acetyl binding and catalysis. Further research on the acetyl binding pocket will provide insights into substrate specificity and aid in efficient biocatalyst development for industrial applications.

Sesame oil downregulates the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis

Atherosclerosis is a chronic inflammatory disease forming plaques in medium and large-sized arteries. ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) is an extracellular-matrix remodelling enzyme involved in the degradation of versican in the arterial wall. Recent reports indicated that increased expression of ADAMTS-4 is associated with plaque progression and vulnerability. Bioactive components of dietary oil, like sesame oil, are reported to have anti-inflammatory and antioxidant properties. Here, we studied the effect of sesame oil on regulating ADAMTS-4 in high-fat diet-induced atherosclerosis rat model. Our results indicated that sesame oil supplementation improved the anti-inflammatory and anti-oxidative status of the body. It also reduced atherosclerotic plaque formation in high-fat diet-fed rats. Our results showed that the sesame oil supplementation significantly down-regulated the expression of ADAMTS-4 in serum and aortic samples. The versican, the large proteoglycan substrate of ADAMTS-4 in the aorta, was downregulated to normal control level on sesame oil supplementation. This study, for the first time, reveals that sesame oil could down-regulate the expression of ADAMTS-4 in high-fat diet-induced atherosclerosis, imparting a new therapeutic potential for sesame oil in the management of atherosclerosis.

In silico mining and identification of a novel lipase from Paenibacillus larvae: Rational protein design for improving catalytic performance

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

P‐107: High‐Resolution Periodic Patterning for Volume Manufacturing on +300mm Size Substrates Using Displacement Talbot Lithography

The photonic device market has a need for low‐cost, high‐resolution periodic patterning that scales with product maturity to large substrate sizes. Eulitha's DTL (Displacement Talbot Lithography) technology was limited to only a single exposure field per substrate on preexisting platforms which can fully pattern 100 and 150mm substrates. The new ‘PhableS' is Eulitha's step‐and‐repeat solution to bring all the advantages of the DTL patterning to larger substrates.The PhableS tool builds on Eulitha's patented displacement Talbot lithography technology to open the way for customers to fully scale to high volume production. This tool retains the strong technical performance; including 60nm half pitch resolution (DUV version), 140x140mm2 single shot exposure field, large depth of focus (hundreds of microns), high throughput (+40wph), non‐contact, mask‐based patterning. Automated wafer and mask handling support industry expected repeatability and defect‐free patterning.

Breaking 1.7 V Open Circuit Voltage in Large Area Transparent Perovskite Solar Cells Using Interfaces Passivation

AbstractEfficient semi‐transparent solar cells can extend the adoption of photovoltaics beyond standard utility‐scale, commercial, or residential applications. Halide perovskites are particularly suitable in this respect owing to their tunable bandgap. The main drawbacks in the development of transparent perovskite solar cells are the high open‐circuit voltage (Voc) deficit and the difficulties in depositing high‐quality thin films over large area substrates, given the low solubility of bromide and chloride precursors. In this work, passivation strategies are developed for the high bandgap Br perovskite able to reduce charge recombination and consequently improve the Voc. The study demonstrates 1 cm2 perovskite solar cells with Voc up to 1.73 V (1.83 eV Quasi Fermi Level Splitting) and a PCE of 8.1%. The average visible transmittance (AVT) exceeds 70% by means of a bifacial light management and a record light utilization efficiency (LUE) of 5.72 is achieved. Moreover, the potential use of the technology is evaluated toward Internet of Things (IoT) application owing to a bifaciality factor of 87% along with 17% PCE under indoor lighting. Finally, the up‐scaling is demonstrated by fabricating 20 cm2 active area modules with PCE of 7.3% and Voc per cell up to 1.65 V.

Optimizing the Thickness Uniformity of Magnetron Sputtering Deposited Films on a Large-Scale Curved Workpiece Surface

This study prepared magnetron-sputtering deposition film on a large-scale curved workpiece with a favorable thickness uniformity using a rectangular target cathode. For a substrate rotation structure with eccentric rotation/revolution composite motion, the geometrical model of the thickness distribution of the coating film on a large substrate was established in combination with the principle of rectangular cathode magnetron sputtering. The integral formula of the film thickness was derived, and various film thickness distribution patterns under different parameters were simulated. According to the present research results, the film thickness distribution can be optimized by adjusting the revolution-to-rotation radius and eccentric ratios. At an eccentric distance of 400 mm and a radius ratio of 2.78, the most favorable uniformity degree of 0.87 was obtained, which was controlled by the motion path of the fixed point on the substrate. The proposed approach helps to obtain large-scale films with favorable uniformity on the substrate.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS’s steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Window[1]resorcin[3]arenes: A Novel Macrocycle Able to Self-Assemble to a Catalytically Active Hexameric Cage.

The hexameric resorcin[4]arene capsule has been utilized as one of the most versatile supramolecular capsule catalysts. Enlarging its size would enable expansion of the substrate size scope. However, no larger catalytically active versions have been reported. Herein, we introduce a novel class of macrocycles, named window[1]resorcin[3]arene (wRS), that assemble to a cage-like hexameric host. The new host was studied by NMR, encapsulation experiments, and molecular dynamics simulations. The cage is able to bind tetraalkylammonium ions that are too large for encapsulation inside the hexameric resorcin[4]arene capsule. Most importantly, it retained its catalytic activity, and the accelerated conversion of a large substrate that does not fit the closed hexameric resorcin[4]arene capsule was observed. Thus, it will help to expand the limited substrate size scope of the closed hexameric resorcin[4]arene capsule.

Facile one-pot green synthesis of Aloe vera mediated silver nanoparticles and their catalytic efficiency towards organic transformations

A facile approach for the synthesis of Aloe vera mediated silver nano-particles (AgNPs) has been carried out via chemical route. The developed Aloe vera mediated AgNPs were characterized by using various techniques such as SEM, FT-IR etc. Additionally, catalytic applications of this interesting nanomaterial were utilized for the synthesis of 4-phenylthiazole- 2-amine and its derivatives. After completion of catalytic reactions, Aloe vera mediated AgNPs were easily recycled without any significant loss in catalytic activity. The robust features of the current approach involve an outstanding catalytic performance, ease of catalyst retrievability, easy work up procedure, and large substrate tolerance. Besides this, the green chemistry metrics for the products formed such as E-factor (Environment Factor), PMI (Process Mass Intensity), RME (Reaction Mass Efficiency), Atom Economy (AE) and carbon efficiency (CE) were in associated with high efficiency and selectivity which further confirms the effectiveness of this approach. This work highlights the significance of using economic and eco-friendly approach for synthesizing thiazole derivatives in good to excellent yields via Aloe vera mediated AgNPs under waste free conditions.

Universal Glycosyltransferase Continuous Assay for Uniform Kinetics and Inhibition Database Development and Mechanistic Studies Illustrated on ST3GAL1, C1GALT1, and FUT1.

Chemical systems glycobiology requires experimental and computational tools to make possible big data analytics benefiting genomics and proteomics. The impediment to tool development is that the nature of glycan construction and mutation is not template driven but rests on cooperative glycosyltransferase (GT) catalytic synthesis. What is needed is the collation of kinetics and inhibition data in a standardized form to make possible analytics of glycan and glycoconjugate synthesis, mechanism extraction, and pattern recognition. Currently, kinetics assays in use for GTs are not universal in processing nucleoside phosphate UDP, GDP, and CMP donor-based glycosylation reactions due to limitations in accuracy and large substrate volume requirements. Here we present a universal glycosyltransferase continuous (UGC) assay able to measure the declining concentration of the NADH reporter molecule through fluorescence spectrophotometry and, therefore, determine reaction rate parameters. The development and parametrization of the assay is based on coupling the nucleotide released from GT reactions with pyruvate kinase, via nucleoside diphosphate kinase (NDK) in the case of NDP-based donor reactions. In the case of CMP-based reactions, the coupling is carried out via another kinase, cytidylate kinase in combination with NDK, which phosphorylates CMP to CDP, then CDP to CTP. Following this, we conduct kinetics and inhibition assay studies on the UDP, GDP, and CMP-based glycosylation reactions, specifically C1GAlT1, FUT1, and ST3GAL1, to represent each class of donor, respectively. The accuracy of calculating initial rates using the continuous assay compared to end point (noncontinuous) assays is demonstrated for the three classes of GTs. The previously identified natural product soyasaponin1 inhibitor was used as a model to demonstrate the application of the UGC assay as a standardized inhibition assay for GTs. We show that the dose response of ST3GAL1 to a serial dilution of Soyasaponin1 has time-dependent inhibition. This brings into question previous inhibition findings, arrived at using an end point assay, that have selected a seemingly random time point to measure inhibition. Consequently, using standardized Km values taken from the UGC assay study, ST3GAL1 was shown to be the most responsive enzyme to soyasaponin1 inhibition, followed by FUT1, then C1GALT1 with IC50 values of 37, 52, and 886 μM respectively.

Broadband Photodetection of Centimeter-Scale T-Phase Gallium Telluride Grown by Molecular Beam Epitaxy.

Two-dimensional (2D) semiconductors have recently attracted considerable attention due to their promising applications in future integrated electronic and optoelectronic devices. Large-scale synthesis of high-quality 2D semiconductors is an increasingly essential requirement for practical applications, such as sensing, imaging, and communications. In this work, homogeneous 2D GaTe films on a centimeter scale are epitaxially grown on fluorphlogopite mica substrates by molecular beam epitaxy (MBE). The epitaxial GaTe thin films showed an atomically 2D layered lattice structure with a T phase, which has not been discovered in the GaTe geometric isomer. Furthermore, semiconducting behavior and high mobility above room temperature were found in T-GaTe epitaxial films, which are essential for application in semiconducting devices. The T-GaTe-based photodetectors demonstrated respectable photodetection performance with a responsivity of 13 mA/W and a fast response speed. By introducing monolayer graphene as the substrate, we successfully realized high-quality GaTe/graphene heterostructures. The performance has been significantly improved, such as the responsivity was enhanced more than 20 times. These results highlight a feasible scheme for exploring the crystal phase of 2D GaTe and realizing the controlled growth of GaTe films on large substrates, which could promote the development of broadband, high-performance, and large-scale photodetection applications.

Innovative syntheses of immobilized CuxO semiconductors grown on 3D prints and their photoelectrochemical activity for sulfamethoxazole degradation

Traditional copper oxide manufacturing processes require high energy consumption, high synthesis temperatures, and long process times. This study proposes an innovative additive manufacturing process to support immobilized copper oxide, offering greater design flexibility and customization. CuxO semiconductors were synthesized on 3D-printed substrates via the anodization process and evaluated for their effectiveness in degrading sulfamethoxazole (SMX). XRD analysis of the samples revealed the presence of both CuO and Cu2O crystalline phases, this mixture of oxides helps to expand the absorption range of visible light. The immobilized CuxO semiconductors exhibited band gaps ranging between 1.6 and 1.8 eV. Negative current densities and Mott-Schottky analysis confirmed the nature of the p-type semiconductor. Semiconductors grown by 3D printing demonstrated remarkable degradation capacity for the antibiotic sulfamethoxazole (SMX), achieving 60% degradation after 360 minutes under visible light. Kinetic degradation tends to be a first-order and zero-order model. TOC analysis further revealed the formation of reaction intermediates during SMX degradation, ultimately leading to the mineralization of the contaminant. Based on these results, we report, for the first time CuxO semiconductors grown on large 3D-printed substrates as promising photocatalysts for the degradation of emerging water pollutants. Based on these results, we report, for the first time, that CuxO semiconductors grown on large 3D-printed substrates are promising photocatalysts for the degradation of emerging water contaminants. Furthermore, it should be emphasized that the proposed method is faster and does not require subsequent thermal treatments for its functionalization, making it attractive for various industrial applications.

Distinct Short-Term Response of Intracellular Amino Acids in Saccharomyces cerevisiae and Pichia pastoris to Oxidative and Reductive Stress

Despite being frequently encountered, the effect of oxidative or reductive stress on the intracellular metabolism and the response of the intracellular metabolome of yeasts is severely understudied. Non-conventional yeasts are attracting increasing attention due to their large substrate portfolio of non-canonical pathways as well as their production and secretion of proteins. To understand the effects of both stresses on yeast, the conventional model yeast S. cerevisiae and the non-conventional model yeast P. pastoris were perturbed with 5 mM of hydrogen peroxide for oxidative stress and 20 mM of dithiothreitol for reductive stress in well-defined chemostat cultures at a steady state, and fermentation profiles, intracellular amino acid levels, and intracellular glutathione levels were measured. Although stable profiles of extracellular metabolites were observed, significant changes were measured in intracellular amino acid levels within the first five minutes. Collectively, the amino acids ranged from 0.5 to 400 µmol/gDW, with the most significant increase upon the induction of oxidative stress being seen in cysteine (up to 90%) for S. cerevisiae and in aspartate (up to 80%) for P. pastoris. Upon the induction of reductive stress, asparagine nearly halves in S. cerevisiae, while tryptophan decreases by 60% in P. pastoris. By inspecting the time traces of each amino acid, possible mechanisms of pathway kinetics are speculated. This work furthers our understanding of the response of metabolism to oxidative stress in two model yeasts.

Multi-microscopy characterization of threading dislocations in CVD-grown diamond films

We present site-coincident imaging of a high dislocation density boron-doped chemical vapor deposition-grown homoepitaxial (001) diamond film using electron channeling contrast imaging (ECCI), cathodoluminescence, transmission electron microscopy (TEM), and scanning secondary ion mass spectroscopy (SIMS). With growing interest in large area heteroepitaxial substrates, we show that ECCI is a promising technique to accurately evaluate dislocations in diamond films. We find the electron backscatter yield is sufficient to distinguish individual threading dislocations in ECCI despite the low atomic number of carbon, and we have generally good agreement between dislocations observed in ECCI and TEM of the same region. Importantly, relying on luminescence in the 430 nm dislocation-related A-band alone results in an underestimation of the defect density as we find only 20%–40% of the threading dislocations luminesce. We further show that dislocations do not perturb the spatial uniformity of boron doping measured by SIMS, even when the dislocations are clustered with high density, and we can tentatively rule out strong segregation effects at the dislocations. Our results establish the complementary use of microscopy and microanalysis methods to rapidly characterize and understand the impact of dislocations in diamond thin films.

Engineering flower-shaped hierarchical micromotors on a sustainable biotemplate by teamed boronate affinity-based surface imprinting for effective separation of shikimic acid

Self-propelled nano/micromotors are the frontier of separation materials because they are capable of converting external energy in the surrounding environment into kinetic energy for their autonomous movement. In this work, a bioinspired flower-shaped hierarchical teamed boronate affinity (TBA)-imprinted Pt-free micromotor is introduced based on waste rape pollen as a biotemplate due to its merits of sustainability, rich source and low price. Such micromotor, composed of MnO2 nanosheets as a catalytic medium, Mg-Al layered double hydroxides (LDH) nanosheets as large surface area substrate and functional surface imprinted polymers as identification subject, exhibits autonomic motor behavior powdered by oxygen bubbles generated by the decomposition of H2O2 and selective recognition and separation for shikimic acid (SA). The hierarchical flower-shaped structure affords more accessible recognition sites for SA, which thus facilitates the separation process. Moreover, in combination with the TBA strategy, the micromotor can precisely recognize the target via boronate affinity between boronic acids and cis-diols from SA while showing strong binding capacity under neutral conditions. Benefiting from the pluripotent role of pioneering boronate affinity-covalent imprinted technique, hydrogen bonds interaction and nanoconfinement effect, the resulting affinity towards SA is evidently enhanced with maximal adsorption capacity of 129.51 mg g−1 at neutral pH in the presence of H2O2. After five capture/release cycles, the adsorption capacity remains above 85 %. This proposed flower-shaped hierarchical micromotor expands the scope of potential materials for the adsorption of SA and provides a new and promising direction for fabricating adsorbents applied in the separation and purification of natural products.

Fabrication of self-standing large (111) single crystal diamond using bulk growth of (100) CVD diamond and lift-off process

Diamond (111) plane is superior in impurity doping and quantum spin control to (100) plane, however, there are many technical issues in mass production of the substrate, such as a smaller seed substrate size, low removal rate of polishing, and low growth rate. In this study, a large (111) single crystal diamond substrate of 7 mm × 6 mm was fabricated by growing and processing bulk (100) CVD single crystal. Furthermore, using this as a seed substrate, a (111) self-standing plate was fabricated using the lift-off method. The FWHM of the X-ray rocking curve was 46 arcsec and that of the Raman peak was 2 cm−1. This method can be applied to fabrication of any high index plane such as (113) and can be utilized to fabrication of substrate for electronic and quantum devices, device processing to fabricate vertical structure.

An imine reductase that captures reactive intermediates in the biosynthesis of the indolocarbazole reductasporine

Imine reductases (IREDs) and reductive aminases (RedAms) have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Co-crystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and RedAms to intercept large substrates relevant to industrial applications.