Substrate Binding Mode Research Articles

Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations.

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.

An unusual dual sugar-binding lectin domain controls the substrate specificity of a mucin-type O-glycosyltransferase.

N-acetylgalactosaminyl-transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (α, β, and γ), where α and β can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both α and β in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The α repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the β repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates α and β into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.

Substrate-Dependent Alteration in the C- and O-Prenylation Specificities of Cannabis Prenyltransferase.

CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.

Deciphering the stereo-specific catalytic mechanisms of cis-epoxysuccinate hydrolases producing L(+)-tartaric acid

Microbial epoxide hydrolases, cis-Epoxysuccinate hydrolases (CESHs) have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.

Assessing plasmon-induced reactions by a combined quantum chemical-quantum/classical hybrid approach.

Plasmon-driven reactions on metal nanoparticles feature rich and complex mechanistic contributions, involving a manifold of electronic states, near-field enhancement, and heat, among others. Although localized surface plasmon resonances are believed to initiate these reactions, the complex reactivity demands deeper exploration. This computational study investigates factors influencing chemical processes on plasmonic nanoparticles, exemplified by protonation of 4-mercaptopyridine (4-MPY) on silver nanoparticles. We examine the impact of molecular binding modes and molecule-molecule interactions on the nanoparticle's surface, near-field electromagnetic effects, and charge-transfer phenomena. Two proton sources were considered at ambient conditions, molecular hydrogen and water. Our findings reveal that the substrate's binding mode significantly affects not only the energy barriers governing the thermodynamics and kinetics of the reaction but also determine the directionality of light-driven charge-transfer at the 4-MPY-Ag interface, pivotal in the chemical contribution involved in the reaction mechanism. In addition, significant field enhancement surrounding the adsorbed molecule is observed (eletromagnetic contribution) which was found insufficient to modify the ground state thermodynamics. Instead, it initiates and amplifies light-driven charge-transfer and thus modulates the excited states' reactivity in the plasmonic-molecular hybrid system. This research elucidates protonation mechanisms on silver surfaces, highlighting the role of molecular-surface and molecule-molecule-surface orientation in plasmon-catalysis.

Assessing the role of residue Phe108 of cytochrome P450 3A4 in allosteric effects of midazolam metabolism.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more drugs in clinical use than any other xenobiotic-metabolizing enzyme. CYP3A4-mediated drug metabolism is usually allosterically modulated by substrate concentration (homotropic allostery) and other drugs (heterotropic allostery), exhibiting unusual kinetic profiles and regiospecific metabolism. Recent studies suggest that residue Phe108 (F108) of CYP3A4 may have an important role in drug metabolism. In this work, residue mutations were coupled with well-tempered metadynamics simulations to assess the importance of F108 in the allosteric effects of midazolam metabolism. Comparing the simulation results of the wild-type and mutation systems, we identify that the π-π interaction and steric effect between the F108 side chain and midazolam is favorable for the stable binding of substrate in the active site. F108 also plays an important role in the transition of substrate binding mode, which mainly induces the transition of substrate binding mode by forming π-π interactions with multiple aromatic rings of the substrate. Moreover, the side chain of F108 is closely related to the radius and depth of the 2a and 2f channels, and F108 may further regulate drug metabolism by affecting the pathway, orientation, or time of substrate entry into the CYP3A4 active site or product egress from the active site. Altogether, we suggest that F108 affects drug metabolism and regulatory mechanisms by affecting substrate binding stability, binding mode transition, and channel characteristics of CYP3A4. Our findings could promote the understanding of complicated allosteric mechanisms in CYP3A4-mediated drug metabolism, and the knowledge could be used for drug development and disease treatment.

Structure and function of the pseudouridine 5’-monophosphate glycosylase PUMY from Arabidopsis thaliana

ABSTRACT Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5’-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5’-monophosphate (ΨMP) into uridine and ribose 5’-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5’-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.

Insight into the Inhibitory Mechanism of Embryonic Ectoderm Development Subunit by Triazolopyrimidine Derivatives as Inhibitors through Molecular Dynamics Simulation.

Inhibition of the Embryonic Ectoderm Development (EED) subunit in Polycomb Repressive Complex 2 (PRC2) can inhibit tumor growth. In this paper, we selected six experimentally designed EED competitive Inhibitors of the triazolopyrimidine derivatives class. We investigated the difference in the binding mode of the natural substrate to the Inhibitors and the effects of differences in the parent nuclei, heads, and tails of the Inhibitors on the inhibitory capacity. The results showed that the binding free energy of this class of Inhibitors was close to or lower compared to the natural substrate, providing an energetic basis for competitive inhibition. For the Inhibitors, the presence of a strong negatively charged group at the 6-position of the parent nucleus or the 8'-position of the head would make the hydrogen atom on the head imino group prone to flip, resulting in the vertical movement of the parent nucleus, which significantly decreased the inhibitory ability. When the 6-position of the parent nucleus was a nonpolar group, the parent nucleus would move horizontally, slightly decreasing the inhibitory ability. When the 8'-position of the head was methylene, it formed an intramolecular hydrophobic interaction with the benzene ring on the tail, resulting in a significant increase in inhibition ability.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

Anisotropic dynamics of an interfacial enzyme active site observed using tethered substrate analogs and ultrafast 2D IR spectroscopy.

Enzymes accelerate the rates of biomolecular reactions by many orders of magnitude compared to bulk solution, and it is widely understood that this catalytic effect arises from a combination of polar pre-organization and electrostatic transition state stabilization. A number of recent reports have also implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity. However, complications arising from spatially-distributed disorder, the occurrence of multiple substrate binding modes, and the influence of hydration dynamics on solvent-exposed active sites still confound many experimental studies. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the promiscuous Pyrococcus horikoshii ene-reductase (PhENR) active site in two binding configurations mimicking proposed "inactive" and "reactive" Michaelis complexes. Spectral diffusion measurements of aryl-nitrile substrate analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5ps) motions. Fermi resonant aryl-azide analogs that sense interactions of coupled oscillators are described. Lineshape and quantum beat analyses of these probes reveal characteristics that correlate with aryl-nitrile frequency fluctuation correlation functions parameters, demonstrating that this anisotropy is an intrinsic property of the water-exposed active site, where countervailing gradients of fast dynamics and disorder in the reactant ground state are maintained near the hydration interface. Our results suggest several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR. This study also highlights a strategy to detect perturbations to vibrational modes outside the transparent window of the mid-IR spectrum, which may be extended to other macromolecular systems.

Probing the substrate binding modes and catalytic mechanisms of BLEG-1, a promiscuous B3 metallo-β-lactamase with glyoxalase II properties.

BLEG-1 from Bacillus lehensis G1 is an evolutionary divergent B3 metallo-β-lactamase (MBL) that exhibited both β-lactamase and glyoxalase II (GLXII) activities. Sequence, phylogeny, biochemical and structural relatedness of BLEG-1 to B3 MBL and GLXII suggested BLEG-1 might be an intermediate in the evolutionary path of B3 MBL from GLXII. The unique active site cavity of BLEG-1 that recognizes both β-lactam antibiotics and S-D-lactoylglutathione (SLG) had been postulated as the key factor for its dual activity. In this study, dynamic ensembles of BLEG-1 and its substrate complexes divulged conformational plasticity and binding modes of structurally distinct substrates to the enzyme, providing better insights into its structure-to-function relationship and enzymatic promiscuity. Our results highlight the flexible nature of the active site pocket of BLEG-1, which is governed by concerted loop motions involving loop7+α3+loop8 and loop12 around the catalytic core, thereby moulding the binding pocket and facilitate interactions of BLEG-1 with both ampicillin and SLG. The distribution of (i) predominantly hydrophobic amino acids in the N-terminal domain, and (ii) flexible amino acids with polar and/or charged side chains in both N- and C-termini provide additional advantages to BLEG-1 in confining the aromatic group of ampicillin, and polar groups of SLG, respectively. The importance of these residues for substrates binding was further confirmed by the reduction in MBL and GLXII activities upon alanine substitutions of Ile-10, Phe-57, Arg-94, Leu-95, and Arg-159. Based on molecular dynamics simulation, mutational, and biochemical data presented herein, the catalytic mechanisms of BLEG-1 toward the hydrolysis of β-lactams and SLG were proposed.

Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase enzyme.

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site.

Exploiting natural products to discover multitarget inhibitors of insect chitinolytic enzymes

Multitarget insecticides are more likely to achieve comprehensive regulation of insects with a low risk of resistance. Insect chitinolytic enzymes, including GH18 chitinases and GH20 β-N-acetyl-d-hexosaminidase, are attractive targets for multitarget insecticide design by virtue of their similar substrate binding mode and catalytic mechanism. On the other hand, natural products with diverse skeletal structures are potential repositories of chitinolytic enzyme inhibitors. This minireview first presents the theoretical basis of developing multitargeted inhibitors against insect chitinolytic enzymes and then focuses on the progress in developing multitarget inhibitors derived from natural products. These may provide ideas for the exploitation of novel pesticide leads from natural products by targeting multiple targets.

Mode of substrate binding for ketohexokinase across isozymes and species implies an induced-fit mechanism

Mode of substrate binding for ketohexokinase across isozymes and species implies an induced-fit mechanism

Computer-aided discovery of a novel thermophilic laccase for low-density polyethylene degradation

Polyethylene (PE) and industrial dyes are recalcitrant pollutants calling for the development of sustainable solutions for their degradation. Laccases have been explored for removal of contaminants and pollutants, including dye decolorization and plastic degradation. Here, a novel thermophilic laccase from PE-degrading Lysinibaccillus fusiformis (LfLAC3) was identified through a computer-aided and activity-based screening. Biochemical studies of LfLAC3 indicated its high robustness and catalytic promiscuity. Dye decolorization experiments showed that LfLAC3 was able to degrade all the tested dyes with decolorization percentage from 39% to 70% without the use of a mediator. LfLAC3 was also demonstrated to degrade low-density polyethylene (LDPE) films after eight weeks of incubation with either crude cell lysate or purified enzyme. The formation of a variety of functional groups was detected using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Damage on the surfaces of PE films was observed via scanning electron microscopy (SEM). The potential catalytic mechanism of LfLAC3 was disclosed by structure and substrate-binding modes analysis. These findings demonstrated that LfLAC3 is a promiscuous enzyme that has promising potential for dye decolorization and PE degradation.

The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.

The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.

De Novo Computational Design of a Lipase with Hydrolysis Activity towards Middle-Chained Fatty Acid Esters.

Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the theozyme constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta "inside-out" protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD1 exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of theozyme intact. The redesigned lipase 1a8uD1-M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate-3.34 times higher than that of 1a8uD1. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD1 and the redesigned 1a8uD1-M8 were devised from scratch. More importantly, the designed 1a8uD1-M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions.

On the Binding Mode and Molecular Mechanism of Enzymatic Polyethylene Terephthalate Degradation

Enzymatic degradation of polyethylene terephthlate (PET) by polyester hydrolases is currently subject to intensive research, as it is considered as a potential eco-friendly recycling method for plastic waste. However, the substrate-binding mode and the molecular mechanism of enzymatic PET hydrolysis are still under intense investigation, and controversial hypotheses have been presented. To help unravel the inherent mechanism of biocatalytic PET degradation at the atomic level, we performed solid-state NMR measurements of a cutinase from Thermobifida fusca (TfCut2) embedded in trehalose glasses together with chemically synthesized, amorphous 13C(═O)-labeled oligomeric PET. The resulting ternary enzyme-PET-trehalose glassy system enabled advanced solid-state NMR methods for real-time tracking of the enzymatic PET degradation and the investigation of PET chain dynamics. Combined with enhanced-sampling molecular dynamics simulations, specific enzyme–substrate interactions during the degradation process could also be monitored. Our results demonstrate that the PET chain is first cleaved by TfCut2 in blocks of at least one repeat unit and further to terephthalic acid and ethylene glycol. Moreover, the second step (formation of final hydrolysis products) appears to be rate-limiting in such reactions. The observed dynamic changes and interfacial protein contacts of 13C-labeled PET carbonyl groups suggest that only one PET repeat unit is bound to the enzyme during the degradation process while the rest of the PET chain is only loosely confined to the active site. These results, not accessible by using conventional solution enzyme samples and small nonhydrolyzable substrates, provide a better understanding of the biocatalytic PET degradation mechanism of polyester hydrolases.

The oxidation of cholesterol derivatives by the CYP124 and CYP142 enzymes from Mycobacterium marinum

The CYP124 and CYP142 families of bacterial cytochrome P450 monooxygenases (CYPs), catalyze the oxidation of methyl branched lipids, including cholesterol, as one of the initial activating steps in their catabolism. Both enzymes are reported to supplement the CYP125 family of P450 enzymes. These CYP125 enzymes are found in the same bacteria, and are the primary cholesterol/cholest-4-en-3-one metabolizing enzymes. To further understand the role of the CYP124 and CYP142 cytochrome P450s we investigated the Mycobacterium marinum enzymes, MmarCYP124A1 and CYP142A3, with various cholesterol analogs with modifications on the A and B rings of the steroid. We assessed the substrate binding and catalytic activity of each enzyme. Neither enzyme could bind or oxidize cholesteryl acetate or 3,5-cholestadiene, which have modifications at the C3 hydroxyl moiety of cholesterol. The CYP142 enzyme was better able to accommodate and oxidize cholesterol analogs which have changes on the A/B rings including cholesterol-5α,6α-epoxide and diastereomers of 5-cholestan-3-ol. The CYP124 enzyme was more tolerant of changes at C7 of the cholesterol B ring, e.g., 7-ketocholesterol than in the A ring. The selectivity for oxidation at the ω-carbon of a branched chain was observed in all steroids that were oxidized. The 7-ketocholesterol-bound MmarCYP124A1 enzyme from M. marinum, was structurally characterized by X-ray crystallography to 1.81 Å resolution. The 7-ketocholesterol-bound X-ray crystal structure of the MmarCYP124A1 enzyme revealed that the substrate binding mode of this cholesterol derivative was altered compared to those observed with other non-steroidal ligands. The structure provided an explanation for the selectivity of the enzyme for terminal methyl hydroxylation.

Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.