RESUMEN
The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.
Asunto(s)
Asparaginasa , Asparagina , Animales , Cobayas/metabolismo , Amoníaco/química , Asparaginasa/genética , Asparaginasa/metabolismo , Asparaginasa/uso terapéutico , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico , Mamíferos/metabolismo , MutaciónRESUMEN
Glycosyltransferases (GTs) from the GT1 family are responsible for the glycosylation of various important organic structures such as terpenes, steroids and peptide antibiotics, making it one of the most intensely studied families of GTs. The target of our study, LanGT2, is a member of the GT1 family that uses an inverting mechanism for transferring olivose from TDP-olivose, the donor substrate, to the natural product tetrangulol (Tet), the precursor of the antibiotic landomycin A. X-ray crystallography in conjunction with mutagenesis experiments has revealed the catalytic significance of 3 amino acids (Ser10, Ser219 and Asp137), suggesting Asp137 as the base catalyst. In the absence of X-ray structures that include the acceptor substrate Tet, in silico experiments and MD simulations that have modeled ternary complexes propose that Asp137 could recruit a water molecule to facilitate the nucleophilic activation of Tet, since the distance between Asp137 and the nucleophile is too long to directly deprotonate the nucleophilic moiety. So far, there is no computational evidence regarding the precise mechanism by which LanGT2 catalyzes the transfer of olivose, which raises questions such as: is a water-assisted mechanism possible? and how does this metal ion-independent GT stabilize the growing negative charge of the diphosphate leaving group? In this work, the QM/MM approach was used to unravel the catalytic mechanism of LanGT2, and to identify the role of crucial catalytic amino acids at a molecular level. Our calculations show that the minimum energy path (MEP) describes an SN2-like mechanism, identifying an oxocarbenium ion-like TS in which the olivosyl moiety adopts a 4H3 conformation. Interactions established between the diphosphate group of TDP and Ser10, Ser219, Arg220 and His283 are key to stabilize the development of charge on the leaving group. Our work also suggests that a water-mediated proton transfer mechanism is feasible, in which the water molecule is key to stabilize the phenolate ion-like nucleophile in the TS. This is the first computational insight into the inverting mechanism of an antibiotic natural product GT, and its implications may serve to guide the design of new biocatalysts for natural product glycodiversification.
Asunto(s)
Productos BiológicosRESUMEN
Carbohydrate processing enzymes are of biocatalytic interest. Glycoside hydrolases and the recently discovered lytic polysaccharide monooxygenase for their use in biomass degradation to obtain biofuels or valued chemical entities. Glycosyltransferases or engineered glycosidases and phosphorylases for the synthesis of carbohydrates and glycosylated products. Quantum mechanics-molecular mechanics (QM/MM) methods are highly contributing to establish their different chemical reaction mechanisms. Other computational methods are also used to study enzyme conformational changes, ligand pathways, and processivity, e.g. for processive glycosidases like cellobiohydrolases. There is still a long road to travel to fully understand the role of conformational dynamics in enzyme activity and also to disclose the variety of reaction mechanisms these enzymes employ. Additionally, computational tools for enzyme engineering are beginning to be applied to evaluate substrate specificity or aid in the design of new biocatalysts with increased thermostability or tailored activity, a growing field where molecular modeling is finding its way.
Asunto(s)
Carbohidratos/química , Química Computacional , Enzimas/química , Simulación de Dinámica Molecular , Especificidad por SustratoRESUMEN
Many bioactive compounds are O-glycosylated metabolites; however, the hydrolytic sensitivity of O-glycosidic linkage limits their therapeutic applications. Enzymatically and chemically stable C-glycosidic bonds are thought of as a potential solution to overcome this problem, although the insufficient information about the structural preferences and interactions that distinguish the C- from the O-glycosylation reactions has hindered the development of enzyme engineering strategies. Thus, in this work, the O-glycosyltransferase LanGT2 (O-LanGT2) and its engineered C-C bond-forming variant (C-LanGT2), which catalyze the initial glycosylation step in the biosynthesis of the antibiotic landomycin A, were studied by means of all-atom Molecular Dynamics simulations. Our results indicate that precise positioning of the acceptor substrate tetrangulol (TET) seems to be determined by the flexibility of the loop 51-62, which gives rise to slightly different secondary structural elements that modulate the interactions between this loop and TET. In O-LanGT2, the most notable interactions between TET and the loop 51-62 involve R59 and A62, whereas in C-LanGT2 they involve A8, I58, and I62. Although A8 is not part of the loop 51-62, it turns out to be key to the binding mode exhibited by TET in C-LanGT2. Thus, the TET-A62 (O-LanGT2) and TET-A8 (C-LanGT2) interactions appear to be critical to accomplish the O- and the C-glycosidic bond specificity, respectively. Finally, all results together shed light on the molecular basis governing the O- and C-bond specificity, revealing that the underlying molecular mechanism that tunes the orientation of TET at its binding pocket involves hydrophobic interactions.
Asunto(s)
Aminoglicósidos/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Simulación de Dinámica Molecular , Mutación , Glicosilación , Glicosiltransferasas/genética , Unión Proteica , Conformación ProteicaRESUMEN
Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate-dependent enzyme involved in the biosynthesis of valine, leucine, isoleucine, and lysine. Experimental evidence has shown that mutation of the Gln202 residue results in a decrease in the enzymatic activity, thus suggesting the main role of the carboligation catalyzed by AHAS. It has been postulated that this residue acts as an acid/base group, protonating the carbonyl oxygen from the 2-ketoacid substrate, during the carboligation reaction. However, previous studies have revealed that 2-ketoacid is not engaged in catalytically relevant interactions with ionizable groups that can act as an acid/base group during the catalysis. Therefore, it has been proposed that the carboligation reaction could occur through an intramolecular proton transfer without the assistance of an amino acid residue with acid-base properties. To decipher the role of Gln202, in this work, we studied the last two catalytic steps of the AHAS through quantum mechanics/molecular mechanics calculations using a full enzyme model of the wild-type AHAS and the Gln202Ala mutant. Our results indicate that the carboligation mechanism occurs through an intramolecular proton transfer that does not require the action of an additional acid-base group. The mechanism is composed of two steps in which the last one is rate-limiting. Our findings reveal that Gln202 stabilizes a catalytic water molecule in the reactive site through electrostatic contributions that are mostly relevant during the carboligation step, in agreement with experimental evidence. The catalytic water engages in intermolecular hydrogen bonds with the reacting species and makes a strong electronic contribution to the stabilization of the reaction intermediate (AL-ThDP).
Asunto(s)
Acetolactato Sintasa/química , Acetolactato Sintasa/metabolismo , Biocatálisis , Glutamina , Levaduras/enzimología , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Teoría CuánticaRESUMEN
In this work, molecular dynamics and QM/MM calculations were employed to examine the structural and catalytic features of the retaining glucosyltransferase GTF-SI from the GH70 family, which participates in the process of caries formation. Our goal was to obtain a deeper understanding of the role of R475 in the mechanism of sucrose breakage. This residue is highly conserved in the GH70 family and so far there has been no evidence that shows what could be the role of this residue in the catalysis performed by GTF-SI. In order to understand the structural role of R475 in the native enzyme, we built full enzyme models of the wild type and the mutants R475A and R475Q. These models were addressed by means of molecular dynamics simulations, which allowed the assessment of the dynamical effect of the R475 mutation on the active site. Then, representative structures were chosen for each one of the mutant models and QM/MM calculations were carried out to unravel the catalytic role of R475. Our results show that the R475 mutation increases the flexibility of the enzyme, which triggers the entrance of water molecules in the active site. In addition, QM/MM calculations indicate that R475 is able to provide a great stabilization to the carboxylate moiety of the acid/base E515, which is an essential characteristic favoring the proton transfer process that promotes the glycosidic bond breakage of sucrose.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Streptococcus mutans/enzimología , Arginina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Dominio Catalítico , Glucosiltransferasas/química , Glucosiltransferasas/genética , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Teoría Cuántica , Sacarosa/química , Sacarosa/metabolismoRESUMEN
Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.
Asunto(s)
Dominio Catalítico , Glucosidasas/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Biocatálisis , Cristalografía por Rayos X , Pruebas de Enzimas/métodos , Glucosidasas/química , Glucosidasas/aislamiento & purificación , Glicósidos/metabolismo , Hordeum/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Plantones/metabolismo , Especificidad por SustratoRESUMEN
Glucansucrase GTF-SI from Streptococcus mutans is a multidomain enzyme that catalyzes the synthesis of glucan polymers. Domain V locates 100 Å from the catalytic site and is required for an optimal activity. Nevertheless, the mechanism governing its functional role remains elusive. In this work, homology modeling and molecular dynamics simulations were employed to examine the effect of domain V in the structure and glucan-binding ability of GTF-SI in full and truncated enzyme models. Our results showed that domain V increases the flexibility of the α4'-loop-α4â³ motif near the catalytic site resulting in a higher surface for glucan association, and modulates the orientation of a growing oligosaccharide (N=8-23) in glucan-enzyme complexes towards engaging in favorable contacts throughout the protein, whereas in the truncated model the glucan protrudes randomly from domain B towards the solvent. These results are valuable to increase understanding about the functional role of domain V in GH70 glucansucrases.
Asunto(s)
Glucanos/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Streptococcus mutans/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Homología de SecuenciaRESUMEN
In this work, QM/MM calculations were employed to examine the catalytic mechanism of the retaining glucosyltransferase GTF-SI enzyme, which participates in the process of caries formation. Our goal was to characterize, with atomistic details, the mechanism of sucrose hydrolysis and the catalytic factors that modulate this reaction. Our results suggest a concerted mechanism for sucrose hydrolysis in which the first event corresponds to the glycosidic bond breakage assisted by Glu515, followed by the nucleophilic attack of Asp477, leading to the formation of the Covalent Glycosyl Enzyme (CGE) intermediate. A novel conformational itinerary of the glucosyl moiety along the reaction mechanism was identified: 2H3 â 2H3-E3 â 4C1, and the calculated energy barrier is 16.4 kcal mol-1, which is in good agreement with experimental evidence showing a major contribution coming from the glycosidic bond breakage. Our calculations also revealed that Arg475 and Asp588 play a critical role as TS-stabilizers by electrostatic and charge transfer mechanisms, respectively. This is the first report dealing with the specific features of the mechanism and catalytic residues involved in GTF-SI hydrolysis of sucrose, which is a matter of relevance in enzyme catalysis and could be valuable to aid the design of novel and specific inhibitors targeting GTF-SI.
Asunto(s)
Proteínas Bacterianas/química , Glucosiltransferasas/química , Streptococcus mutans/enzimología , Catálisis , Glicosilación , Hidrólisis , Modelos Moleculares , Teoría Cuántica , Sacarosa/química , TermodinámicaRESUMEN
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.
Asunto(s)
Dominio Catalítico , N-Acetilglucosaminiltransferasas/metabolismo , Glicosaminoglicanos/metabolismo , Simulación de Dinámica Molecular , Mutación , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Estereoisomerismo , Especificidad por SustratoRESUMEN
Glycosyltransferases (GTs) catalyze the biosynthesis of glycosidic linkages by transferring a monosaccharide from a nucleotide sugar donor to an acceptor substrate, and they do that with exquisite regio- and stereospecificity. Retaining GTs act with retention of the configuration at the anomeric carbon of the transferred sugar. Their chemical mechanism has been under debate for long as conclusive experimental data to confirm the mechanism have been elusive. In the past years, quantum mechanical/molecular mechanical (QM/MM) calculations have shed light on the mechanistic discussion. Here, we review the work carried out in our group investigating three of these retaining enzymes (LgtC, α3GalT, and GalNAc-T2). Our results support the controversial front-side attack mechanism as the general mechanism for most retaining GTs. The latest structural data are in agreement with these findings. QM/MM calculations have revealed how enzyme-substrate and substrate-substrate interactions modulate the transfer reaction catalyzed by these enzymes. Moreover, they provide an explanation on why in some cases a strong nucleophilic residue is found on the ß-face of the sugar, opening the door to a shift toward a double-displacement mechanism.
Asunto(s)
Proteínas Bacterianas/química , Galactosiltransferasas/química , Glicosiltransferasas/química , Monosacáridos/química , N-Acetilgalactosaminiltransferasas/química , Bacterias/química , Bacterias/enzimología , Biocatálisis , Humanos , Hidroxilación , Cinética , Simulación de Dinámica Molecular , Teoría Cuántica , Estereoisomerismo , Especificidad por Sustrato , Termodinámica , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
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.