Getting Deeper into the Molecular Events of Heme Binding Mechanisms: A Comparative Multi-level Computational Study of HasAsm and HasAyp Hemophores.

Many biological systems obtain their activity by the inclusion of metalloporphyrins into one or several binding pockets. However, decoding the molecular mechanism under which these compounds bind to their receptors is something that has not been widely explored and is a field with open questions. In the present work, we apply computational techniques to unravel and compare the mechanisms of two heme-binding systems, concretely the HasA hemophores from Gram negative bacteria Serratiamarcescens (HasAsm) and Yersinia pestis (HasAyp). Despite the high sequence identity between both systems, the comparison between the X-ray structures of their apo and holo forms suggests different heme-binding mechanisms. HasAyp has extremely similar structures for heme-free and heme-bound forms, while HasAsm presents a very large displacement of a loop that ultimately leads to an additional coordination to the metal with respect to HasAyp. We combined Gaussian accelerated molecular dynamics simulations (GaMDs) in explicit solvent and protein-ligand docking optimized for metalloligands. GaMDs were first carried out on heme-free forms of both hemophores. Then, protein-ligand dockings of the heme were performed on cluster representatives of these simulations and the best poses were then subjected to a new series of GaMDs. A series of analyses reveal the following: (1) HasAyp has a conformational landscape extremely similar between heme-bound and unbound states with no to limited impact on the binding of the cofactor, (2) HasAsm presents as a slightly broader conformational landscape in its apo state but can only visit conformations similar to the X-ray of the holo form when the heme has been bound. Such behavior results from a complex cascade of changes in interactions that spread from the heme-binding pocket to the flexible loop previously mentioned. This study sheds light on the diversity of molecular mechanisms of heme-binding and discusses the weight between the pre-organization of the receptor as well as the induced motions resulting in association.

Accounting for the instantaneous disorder in the enzyme-substrate Michaelis complex to calculate the Gibbs free energy barrier of an enzyme reaction.

Many enzyme reactions present instantaneous disorder. These dynamic fluctuations in the enzyme-substrate Michaelis complexes generate a wide range of energy barriers that cannot be experimentally observed, but that determine the measured kinetics of the reaction. These individual energy barriers can be calculated using QM/MM methods, but then the problem is how to deal with this dispersion of energy barriers to provide kinetic information. So far, the most usual procedure has implied the so-called exponential average of the energy barriers. In this paper, we discuss the foundations of this method, and we use the free energy perturbation theory to derive an alternative equation to get the Gibbs free energy barrier of the enzyme reaction. In addition, we propose a practical way to implement it. We have chosen four enzyme reactions as examples. In particular, we have studied the hydrolysis of a glycosidic bond catalyzed by the enzyme Thermus thermophilus ß-glycosidase, and the mutant Y284P Ttb-gly, and the hydrogen abstraction reactions from C13 and C7 of arachidonic acid catalyzed by the enzyme rabbit 15-lipoxygenase-1.

Evolutionary alteration of ALOX15 specificity optimizes the biosynthesis of antiinflammatory and proresolving lipoxins.

ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratLeu353Phe) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P < 0.01) [corrected]. Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory and proresolving lipoxins.

Computational insights into active site shaping for substrate specificity and reaction regioselectivity in the EXTL2 retaining glycosyltransferase.

Glycosyltransferases are enzymes that catalyze a monosaccharide transfer reaction from a donor to an acceptor substrate with the synthesis of a new glycosidic bond. They are highly substrate specific and regioselective, even though the acceptor substrate often presents multiple reactive groups. Currently, many efforts are dedicated to the development of biocatalysts for glycan synthesis and, therefore, a better understanding of how natural enzymes achieve this goal can be of valuable help. To gain a deeper insight into the catalytic strategies used by retaining glycosyltransferases, the wild type EXTL2 (CAZy family GT64) and four mutant forms (at positions 293 and 246) were studied using QM(DFT)/MM calculations and molecular dynamics simulations. Existing hypotheses on the roles of Arg293, an enigmatic residue in the CAZy family GT64 that seemed to contradict a mechanism through an oxocarbenium intermediate, and of Asp246 have been tested. We also provide a molecular interpretation for the results of site-directed mutagenesis experiments. Moreover, we have investigated why an Asp, and not a Glu like in the family GT6, is found on the ß-face of the transferred GlcNAc. It is predicted that an Asp246Glu mutant of EXTL2 would be unable to catalyze the α-1,4 transfer. The results herein presented clarify the roles that Arg293, Asp246 and Leu213 have at different stages of the catalytic process (for binding but also for efficient chemical reaction). Altogether, we provide a molecular view that connects the identity and conformation of these residues to the substrate specificity and regioselectivity of the enzyme, illustrating a delicate interplay between all these aspects.

The importance of ensemble averaging in enzyme kinetics.

CONSPECTUS: The active site of an enzyme is surrounded by a fluctuating environment of protein and solvent conformational states, and a realistic calculation of chemical reaction rates and kinetic isotope effects of enzyme-catalyzed reactions must take account of this environmental diversity. Ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT) was developed as a way to carry out such calculations. This theory incorporates ensemble averaging, quantized vibrational energies, energy, tunneling, and recrossing of transition state dividing surfaces in a systematic way. It has been applied successfully to a number of hydrogen-, proton-, and hydride-transfer reactions. The theory also exposes the set of effects that should be considered in reliable rate constants calculations. We first review the basic theory and the steps in the calculation. A key role is played by the generalized free energy of activation profile, which is obtained by quantizing the classical potential of mean force as a function of a reaction coordinate because the one-way flux through the transition state dividing surface can be written in terms of the generalized free energy of activation. A recrossing transmission coefficient accounts for the difference between the one-way flux through the chosen transition state dividing surface and the net flux, and a tunneling transmission coefficient converts classical motion along the reaction coordinate to quantum mechanical motion. The tunneling calculation is multidimensional, accounting for the change in vibrational frequencies along the tunneling path and shortening of the tunneling path with respect to the minimum energy path (MEP), as promoted by reaction-path curvature. The generalized free energy of activation and the transmission coefficients both involve averaging over an ensemble of reaction paths and conformations, and this includes the coupling of protein motions to the rearrangement of chemical bonds in a statistical mechanically correct way. The standard deviations of the transmissions coefficients provide information on the diversity of the distribution of reaction paths, barriers, and protein conformations along the members of an ensemble of reaction paths passing through the transition state. We first illustrate the theory by discussing the application to both wild-type and mutant Escherichia coli dihydrofolate reductase and hyperthermophilic Thermotoga maritima dihydrofolate reductase (DHFR); DHFR is of special interest because the protein conformational changes have been widely studied. Then we present shorter discussions of several other applications of EA-VTST/MT to transfer of protons, hydrogen atoms, and hydride ions and their deuterated analogs. Systems discussed include hydride transfer in alcohol dehydrogenase, xylose isomerase, and thymidylate synthase, proton transfer in methylamine dehydrogenase, hydrogen atom transfer in methylmalonyl-CoA mutase, and nucleophilic substitution in haloalkane dehalogenase and two-dimensional potentials of mean force for potentially coupled proton and hydride transfer in the ß-oxidation of butyryl-coenzyme A catalyzed by short-chain acyl-CoA dehydrogenase and in the pyruvate to lactate transformation catalyzed by lactate dehydrogenase.

Is Regioselectivity in the Enzyme-Catalyzed Hydroperoxidation of Arachidonic Acid Necessarily Determined by Hydrogen Abstraction? The Case of Rabbit Leu597Ala/Ile663Ala ALOX15 Mutant.

Molecular dynamics simulations and quantum mechanics/molecular mechanics calculations were performed on the in silico Leu597Ala/Ile663Ala double mutant of rabbit ALOX15 (12/15 lipoxygenase). The computational results suggested that subtle steric hindrance by the conserved Leu597 and C-terminal Ile663 residues disturbed H10 abstractions in wildtype ALOX15 (which abstracts H13), but if these two bulky residues were mutated to smaller ones, H10 abstraction was no longer impeded and the regioselectivity of the initial H-abstraction step was changed. However, site-directed mutagenesis with HPLC analysis of the products of the whole oxidation process showed that the regioselectivity of the hydroperoxidation was not altered. This disagreement may be explained by the conformational reorganization of the system needed to rotate the -OO. group from an antarafacial to a suprafacial arrangement prior to back-hydrogen transfer. After H10 abstraction and O2 insertion, the evolution of the peroxy radical at C12 was sterically impeded, whereas peroxyl group rotation at C15 (after H13 abstraction) could easily evolve to a suprafacial arrangement, which thus led to the final product. For this reason, the global regiospecificity was not affected in the mutant. These findings exemplify that the regioselectivity of initial hydrogen abstraction and the regioselectivity of the final product do not necessarily coincide (in fact, they can be opposite) for the hydroperoxidation of arachidonic acid catalyzed by a lipoxygenase.

Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase: consequences for the positional specificity of oxygenation.

In the present work we have combined homology modeling, protein-ligand dockings, quantum mechanics/molecular mechanics calculations and molecular dynamics simulations to generate human 5-lipoxygenase (5-LOX):arachidonic acid (AA) complexes consistent with the 5-lipoxygenating activity (which implies hydrogen abstraction at the C7 position). Our results suggest that both the holo and the apo forms of human Stable 5-LOX could accommodate AA in a productive form for 5-lipoxygenation. The former, in a tail-first orientation, with the AA carboxylate end interacting with Lys409, gives the desired structures with C7 close to the Fe-OH(-) cofactor and suitable barrier heights for H7 abstraction. Only when using the apo form structure, a head-first orientation with the AA carboxylate close to His600 (a residue recently proposed as essential for AA positioning) is obtained in the docking calculations. However, the calculated barrier heights for this head-first orientation are in principle consistent with 5-LOX specificity, but also with 12/8 regioselectivity. Finally, long MD simulations give support to the recent hypothesis that the Phe177 + Tyr181 pair needs to close the active site access during the chemical reaction, and suggest that in the case of a head-first orientation Phe177 may be the residue interacting with the AA carboxylate.

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase.

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host-pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum-mechanics/molecular-mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front-side substrate-assisted SN i-type reaction.

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase.

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme-substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.

Introducing mutations to modify the C13/C9 ratio in linoleic acid oxygenations catalyzed by rabbit 15-lipoxygenase: a QM/MM and MD study.

Lipoxygenases (LOs) are a family of nonheme iron-containing enzymes that catalyze the hydroperoxidation of several polyunsaturated fatty acids with a huge regio- and stereospecificity. Mammalian 15-LO-1 yields almost exclusively oxygenation at the C13 position of the linoleic acid (LA), its preferred substrate. This is very important because metabolites derived from oxidation in distinct positions produce opposite physiological effects. We have combined here quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to show how a suitable mutation of the rabbit 15-LO-1 enzyme can produce a significant amount of products derived from oxygenation at the C9 position of LA. In effect, the Leu597Val or Leu597Ala mutants are predicted to lead to a diminution of the oxygenation C13/C9 ratio in LA as huge as five orders of magnitude. This shows that the conserved residue Leu597 actually drives the regiospecific hydroperoxidation of LA catalyzed by 15-LO-1 enzyme.

Regio- and stereospecificity in the oxygenation of arachidonic acid catalyzed by Leu597 mutants of rabbit 15-lipoxygenase: a QM/MM study.

We combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to study the addition of O2 to the pentadienyl radical of arachidonic acid (AA) catalyzed by the Leu597Val and Leu597Ala mutants of rabbit 15-lipoxygenase (15-rLO). In the Leu597Val mutant, the addition of O2 to C15 of AA is the predominant path, although it reduces the C15/C11 product ratio by almost ten times with respect to the wildtype enzyme. The S stereochemistry is kept. Mutation to Ala causes just the opposite effect: regiospecificity favoring addition to C15 is somewhat sharper than that in the wildtype, but the stereochemistry is R. This is because the extra space created by the mutation to Ala is big enough for AA to move so that it can adopt an alternative binding mode, and this opens new feasible paths for the attack of O2 . So, we showed that the Leu597Ala mutant of 15r-LO works as an aspirin-acetylated cyclooxygenase-2, which makes 15-(R)- hydroperoxyeicosatetraenoic acid.

A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2.

It is estimated that >50% of proteins are glycosylated with sugar tags that can modulate protein activity through what has been called the sugar code. Here we present the first QM/MM calculations of human GalNAc-T2, a retaining glycosyltransferase, which initiates the biosynthesis of mucin-type O-glycans. Importantly, we have characterized a hydrogen bond between the ß-phosphate of UDP and the backbone amide group from the Thr7 of the sugar acceptor (EA2 peptide) that promotes catalysis and that we propose could be a general catalytic strategy used in peptide O-glycosylation by retaining glycosyltransferases. Additional important substrate-substrate interactions have been identified, for example, between the ß-phosphate of UDP with the attacking hydroxyl group from the acceptor substrate and with the substituent at the C2' position of the transferred sugar. Our results support a front-side attack mechanism for this enzyme, with a barrier height of ~20 kcal mol(-1) at the QM(M05-2X/TZVP//BP86/SVP)/CHARMM22 level, in reasonable agreement with the experimental kinetic data. Experimental and in silico mutations show that transferase activity is very sensitive to changes in residues Glu334, Asn335 and Arg362. Additionally, our calculations for different donor substrates suggest that human GalNAc-T2 would be inactive if 2'-deoxy-Gal or 2'-oxymethyl-Gal were used, while UDP-Gal is confirmed as a valid sugar donor. Finally, the analysis herein presented highlights that both the substrate-substrate and the enzyme-substrate interactions are mainly concentrated on stabilizing the negative charge developing at the UDP leaving group as the transition state is approached, identifying this as a key aspect of retaining glycosyltransferases catalysis.

Substrate-assisted and nucleophilically assisted catalysis in bovine α1,3-galactosyltransferase. Mechanistic implications for retaining glycosyltransferases.

Glycosyltransferases (GTs) are responsible for the biosynthesis of glycans, the most abundant organic molecules in nature. Their biological relevance makes necessary the knowledge of their catalytic mechanism, which in the case of retaining GTs is still a matter of debate. After the initial proposal of a double-displacement mechanism with formation of a covalent glycosyl-enzyme intermediate (CGE), new experimental and computational data are pointing out to a front-side attack as a plausible alternative. The question is then why family GT6 members, like bovine α1,3-galactosyltransferase (α1,3-GalT), have a nucleophilic residue (Glu317) situated close to the anomeric carbon. To answer this and other questions, QM(DFT)/MM calculations on the entire α1,3-GalT:substrates system (and for the E317A/E317Q mutants) have been carried out. We describe a substrate-assisted mechanism for retaining GTs consisting of the stabilization of the developing negative charge on the ß-phosphate by the hydrogen of the attacking hydroxyl group of the acceptor molecule. This interaction is impaired in the α1,3-GalT reactants, which explains why Glu317 is required to nucleophilically assist initial catalysis by "pushing" leaving-group departure. The presence of Glu317 opens the door to the possibility of a double-displacement mechanism in GT6 family. Our results suggest that in α1,3-GalT the substrate-assisted catalysis would be necessary in both mechanisms (for which we predict similar reaction rates), because the nucleophilic strength of Glu317 is reduced by the interactions it makes to ensure proper acceptor binding. Interestingly, the same effect would be found in the absence of the acceptor when Glu317 interacts with water molecules, which could explain the difficulties for isolating the CGE experimentally, and could be a strategy to avoid undesired hydrolysis of the donor substrate.

On the regio- and stereospecificity of arachidonic acid peroxidation catalyzed by mammalian 15-lypoxygenases: a combined molecular dynamics and QM/MM study.

15-Lipoxygenases (15-LOs) catalyse the peroxidation reaction of arachidonic acid (AA) in mammals with remarkable regio- and stereospecificity. This positional-specific peroxidation is of paramount importance because it determines the nature and biological functions of the final metabolites generated by each LO as a result of the oxidative metabolism of AA. Although several hypotheses have been formulated concerning the regio- and stereospecificity of LOs, the molecular basis of such behaviour is still unclear. Herein, we combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations of the complete rabbit 15-LO/AA solvated model to examine the most accepted hypotheses for the regio- and stereospecificity of LOs. We have found that the clue to explain this specificity is the oxygen-targeting hypothesis through steric shielding of specific residues (mainly Leu597, Gln548 and Phe175, as well as the AA tail itself). Our deductions are based primarily on the analysis of the energy barrier heights from the oxygen addition reaction profiles.

Corrigendum to "Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase" Comput Struct Biotechnol J, 20 (2022), 3899-3910.

[This corrects the article DOI: 10.1016/j.csbj.2022.07.032.].

Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Ligand-induced formation of transient dimers of mammalian 12/15-lipoxygenase: a key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.

Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion-like transition state.

Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase.

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK a of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications.