Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies.

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Conformational and Electronic Variations in 1,2- and 1,5a-Cyclophellitols and their Impact on Retaining α-Glucosidase Inhibition.

Glycoside hydrolases (glycosidases) take part in myriad biological processes and are important therapeutic targets. Competitive and mechanism-based inhibitors are useful tools to dissect their biological role and comprise a good starting point for drug discovery. The natural product, cyclophellitol, a mechanism-based, covalent and irreversible retaining ß-glucosidase inhibitor has inspired the design of diverse α- and ß-glycosidase inhibitor and activity-based probe scaffolds. Here, we sought to deepen our understanding of the structural and functional requirements of cyclophellitol-type compounds for effective human α-glucosidase inhibition. We synthesized a comprehensive set of α-configured 1,2- and 1,5a-cyclophellitol analogues bearing a variety of electrophilic traps. The inhibitory potency of these compounds was assessed towards both lysosomal and ER retaining α-glucosidases. These studies revealed the 1,5a-cyclophellitols to be the most potent retaining α-glucosidase inhibitors, with the nature of the electrophile determining inhibitory mode of action (covalent or non-covalent). DFT calculations support the ability of the 1,5a-cyclophellitols, but not the 1,2-congeners, to adopt conformations that mimic either the Michaelis complex or transition state of α-glucosidases.

Evolution of Phosphorylase Activity in an Ancestral Glycosyltransferase.

The reconstruction of ancestral sequences can offer a glimpse into the fascinating process of molecular evolution by exposing the adaptive pathways that shape the proteins found in nature today. Here, we track the evolution of the carbohydrate-active enzymes responsible for the synthesis and turnover of mannogen, a critical carbohydrate reserve in Leishmania parasites. Biochemical characterization of resurrected enzymes demonstrated that mannoside phosphorylase activity emerged in an ancestral bacterial mannosyltransferase, and later disappeared in the process of horizontal gene transfer and gene duplication in Leishmania. By shuffling through plausible historical sequence space in an ancestral mannosyltransferase, we found that mannoside phosphorylase activity could be toggled on through various combinations of mutations at positions outside of the active site. Molecular dynamics simulations showed that such mutations can affect loop rigidity and shield the active site from water molecules that disrupt key interactions, allowing α-mannose 1-phosphate to adopt a catalytically productive conformation. These findings highlight the importance of subtle distal mutations in protein evolution and suggest that the vast collection of natural glycosyltransferases may be a promising source of engineering templates for the design of tailored phosphorylases.

ß-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining ß-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.

Trans-cyclosulfamidate mannose-configured cyclitol allows isoform-dependent inhibition of GH47 α-d-mannosidases through a bump-hole strategy.

Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.

Human O-GlcNAcase Uses a Preactivated Boat-skew Substrate Conformation for Catalysis. Evidence from X-ray Crystallography and QM/MM Metadynamics.

Human O-linked ß-N-acetylglucosaminidase (hOGA) is one of the two enzymes involved in nuclear and cytoplasmic protein O-GlcNAcylation, an essential post-translational modification. The enzyme catalyzes the hydrolysis of the GlcNAc-O-(Ser/Thr) glycosidic bonds via anchimeric assistance through the 2-acetamido group of the GlcNAc sugar. However, the conformational itinerary of the GlcNAc ring during catalysis remains unclear. Here we report the crystal structure of wild type hOGA in complex with a nonhydrolyzable glycopeptide substrate and elucidate the full enzyme catalytic mechanism using QM/MM metadynamics. We show that the enzyme can bind the substrate in either a chair- or a boat-like conformation, but only the latter is catalytically competent, leading to the reaction products via 1,4B/1S3 → [4E]‡ → 4C1 and 4C1 → [4E]‡ → 1,4B/1S3 conformational itineraries for the first and second catalytic reaction steps, respectively. Our results reconcile previous experimental observations for human and bacterial OGA and will aid the development of more effective OGA inhibitors for diseases associated with impaired O-GlcNAcylation.

Control of Substrate Conformation by Hydrogen Bonding in a Retaining ß-Endoglycosidase.

Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OH⋅⋅⋅nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.

Molecular basis for bacterial N-glycosylation by a soluble HMW1C-like N-glycosyltransferase.

Soluble HMW1C-like N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.

Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation.

Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid ß-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of Thermoanaerobacterium xylanolyticum ß-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed 4C1 chair at the -1 subsite. Nevertheless, the reaction can still proceed through a 4H3 half-chair transition state, as in classical retaining ß-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a gauche, trans orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.

In silico modelling of the function of disease-related CAZymes.

In silico modelling of proteins comprises a diversity of computational tools aimed to obtain structural, electronic, and/or dynamic information about these biomolecules, capturing mechanistic details that are challenging to experimental approaches, such as elusive enzyme-substrate complexes, short-lived intermediates, and reaction transition states (TS). The present article gives the reader insight on the use of in silico modelling techniques to understand complex catalytic reaction mechanisms of carbohydrate-active enzymes (CAZymes), along with the underlying theory and concepts that are important in this field. We start by introducing the significance of carbohydrates in nature and the enzymes that process them, CAZymes, highlighting the conformational flexibility of their carbohydrate substrates. Three commonly used in silico methods (classical molecular dynamics (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and enhanced sampling techniques) are described for nonexpert readers. Finally, we provide three examples of the application of these methods to unravel the catalytic mechanisms of three disease-related CAZymes: ß-galactocerebrosidase (GALC), responsible for Krabbe disease; α-mannoside ß-1,6-N-acetylglucosaminyltransferase V (MGAT5), involved in cancer; and O-fucosyltransferase 1 (POFUT1), involved in several human diseases such as leukemia and the Dowling-Degos disease.

Glycosidase mechanisms: Sugar conformations and reactivity in endo- and exo-acting enzymes.

The enzymatic breakdown of carbohydrates plays a critical role in several biological events and enables the development of sustainable processes to obtain bioproducts and biofuels. In this scenario, the design of efficient inhibitors for glycosidases that can act as drug targets and the engineering of carbohydrate-active enzymes with tailored catalytic properties is of remarkable importance. To guide rational approaches, it is necessary to elucidate enzyme molecular mechanisms, in particular understanding how the microenvironment modulates the conformational space explored by the substrate. Computer simulations, especially those based on ab initio methods, have provided a suitable atomic description of carbohydrate conformations and catalytic reactions in several glycosidase families. In this review, we will focus on how the active-site topology (pocket or cleft) and mode of cleavage (endo or exo) can affect the catalytic mechanisms adopted by glycosidases, in particular the substrate conformations along the reaction coordinate.

Enzymatic C4-Epimerization of UDP-Glucuronic Acid: Precisely Steered Rotation of a Transient 4-Keto Intermediate for an Inverted Reaction without Decarboxylation.

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.

4-O-Substituted Glucuronic Cyclophellitols are Selective Mechanism-Based Heparanase Inhibitors.

Degradation of the extracellular matrix (ECM) supports tissue integrity and homeostasis, but is also a key factor in cancer metastasis. Heparanase (HPSE) is a mammalian ECM-remodeling enzyme with ß-D-endo-glucuronidase activity overexpressed in several malignancies, and is thought to facilitate tumor growth and metastasis. By this virtue, HPSE is considered an attractive target for the development of cancer therapies, yet to date no HPSE inhibitors have progressed to the clinic. Here we report on the discovery of glucurono-configured cyclitol derivatives featuring simple substituents at the 4-O-position as irreversible HPSE inhibitors. We show that these compounds, unlike glucurono-cyclophellitol, are selective for HPSE over ß-D-exo-glucuronidase (GUSB), also in platelet lysate. The observed selectivity is induced by steric and electrostatic interactions of the substituents at the 4-O-position. Crystallographic analysis supports this rationale for HPSE selectivity, and computer simulations provide insights in the conformational preferences and binding poses of the inhibitors, which we believe are good starting points for the future development of HPSE-targeting antimetastatic cancer drugs.

Phosphorylation disrupts long-distance electron transport in cytochrome c.

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation.

The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases.

In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

How Do Metalloproteins Tame the Fenton Reaction and Utilize •OH Radicals in Constructive Manners?

This Account describes the manner whereby nature controls the Fenton-type reaction of O-O homolysis of hydrogen peroxide and harnesses it to carry out various useful oxidative transformations in metalloenzymes. H2O2 acts as the cosubstrate for the heme-dependent peroxidases, P450BM3, P450SPα, P450BSß, and the P450 decarboxylase OleT, as well as the nonheme enzymes HppE and the copper-dependent lytic polysaccharide monooxygenases (LPMOs). Whereas heme peroxidases use the Poulos-Kraut heterolytic mechanism for H2O2 activation, some heme enzymes prefer the alternative Fenton-type mechanism, which produces •OH radical intermediates. The fate of the •OH radical is controlled by the protein environment, using tight H-bonding networks around H2O2. The so-generated •OH radical is constrained by the surrounding H-bonding interactions, the orientation of which is targeted to perform H-abstraction from the Fe(III)-OH group and thereby leading to the formation of the active species, called Compound I (Cpd I), Por+•Fe(IV)═O, which performs oxidation of the substrate. Alternatively, for the nonheme HppE enzyme, the O-O homolysis catalyzed by the resting state Fe(II) generates an Fe(III)-OH species that effectively constrains the •OH radical species by a tight H-bonding network. The so-formed H-bonded •OH radical acts directly as the oxidant, since it is oriented to perform H-abstraction from the C-H bond of the substrate (S)-2-HPP. The Fenton-type H2O2 activation is strongly suggested by computations to occur also in copper-dependent LPMOs and pMMO. In LPMOs, the Cu(I)-catalyzed O-O homolysis of the H2O2 cosubstrate generates an •OH radical that abstracts a hydrogen atom from Cu(II)-OH and forms thereby the active species of the enzyme, Cu(II)-O•. Such Fenton-type O-O activation can be shared by both the O2-dependent activations of LPMOs and pMMOs, in which the O2 cosubstrate may be reduced to H2O2 by external reductants. Our studies show that, generally, the H2O2 activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since H2O2 is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of H2O2 from the active site, as has been suggested in P450cam and LPMOs, whereas the presence of the substrate seems to play a role in steering a Fenton-type H2O2 activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of H2O2 and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type H2O2 activation mechanism by enzymes differs from the reaction in synthetic systems. In nonenzymatic reactions, the H-bonding networks are quite dynamic and flexible and the reactivity of H2O2 is not strategically constrained as in the enzymatic environment. As such, our Account describes the controlled Fenton-type mechanism in metalloenzymes, and the role of the protein environment in constraining the •OH radical against oxidative damage, while directing it to perform useful oxidative transformations.

Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut.

Mucinases of human gut bacteria cleave peptide bonds in mucins strictly depending on the presence of neighboring O-glycans. The Akkermansia muciniphila AM0627 mucinase cleaves specifically in between contiguous (bis) O-glycans of defined truncated structures, suggesting that this enzyme may recognize clustered O-glycan patches. Here, we report the structure and molecular mechanism of AM0627 in complex with a glycopeptide containing a bis-T (Galß1-3GalNAcα1-O-Ser/Thr) O-glycan, revealing that AM0627 recognizes both the sugar moieties and the peptide sequence. AM0627 exhibits preference for bis-T over bis-Tn (GalNAcα1-O-Ser/Thr) O-glycopeptide substrates, with the first GalNAc residue being essential for cleavage. AM0627 follows a mechanism relying on a nucleophilic water molecule and a catalytic base Glu residue. Structural comparison among mucinases identifies a conserved Tyr engaged in sugar-π interactions in both AM0627 and the Bacteroides thetaiotaomicron BT4244 mucinase as responsible for the common activity of these two mucinases with bis-T/Tn substrates. Our work illustrates how mucinases through tremendous flexibility adapt to the diversity in distribution and patterns of O-glycans on mucins.