Substrate-assisted Catalytic Mechanism Research Articles

Characterization and structural study of a novel β-N-acetylgalactosaminidase from Niabella aurantiaca.

We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified β-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. Whenrecombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-β-d-galactosamine (pNP-β-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal β-sandwich domain and a (β/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering ofunknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinctsubfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may bea β-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted β-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete.

Insight into substrate-assisted catalytic mechanism and stereoselectivity of bifunctional nocardicin thioesterase.

The inversion from L- to D-stereochemistry endows peptides improved bioactivity and enhanced resistance to many proteases and peptidases. To strengthen the biostability and bioavailability of peptide drugs, enzymatic epimerization becomes an important way to incorporate D-amino acid into peptide backbones. Recently, a bifunctional thioesterase NocTE, which is responsible for the epimerization and hydrolysis of the C-terminal (p-hydroxyphenyl)glycine residue of β-lactam antibiotic nocardicin A, exclusively directs to the generation of D-diastereomers. Different from other epimerases, NocTE exhibits unique stereochemical selectivity. Herein, we investigated the catalytic mechanism of NocTE via molecular dynamic (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations. Through structural analyses, two key water molecules around the reaction site were found to serve as proton mediators in epimerization. The structural characteristics inspired us to propose a substrate-assisted mechanism for the epimerization, where multi-step proton transfers were mediated by water molecules and β-lactam ring, and the free energy barrier was calculated to be 20.3kcal/mol. After that, the hydrolysis of D-configured substrate was energetically feasible with the energy barrier of 14.3 kcal/mol. As a comparison, the energy barrier for the direct hydrolysis of L-configured substrate was obtained to be 24.0kcal/mol. Our study provides mechanistic insights into catalytic activities of bifunctional thioesterase NocTE, uncovers more clues to the molecular basis for stereochemical selectivity and paves the way for the directed biosynthesis of novel peptide drugs with various stereostructural characteristics by enzyme rational design.

The structure and molecular dynamics of prolyl oligopeptidase from Microbulbifer arenaceous provide insights into catalytic and regulatory mechanisms.

Prolyl oligopeptidases (POPs) are atypical serine proteases that are unique in their involvement in the maturation and degradation of prolyl-containing peptide hormones and neuropeptides. They are potential pharmaceutical targets for the treatment of several neurodegenerative disorders, such as Alzheimer's disease. In this study, the catalytic and substrate-regulatory mechanisms of a novel bacterial POP from Microbulbifer arenaceous (MaPOP) were investigated. The crystal structure revealed that the catalytic triad of MaPOP was covered by the central tunnel of an unusual β-propeller domain. The tunnel not only provided the sole access to the active site for oligopeptides, but also protected large structured peptides or proteins from accidental proteolysis. The enzyme was able to cleave angiotensin I specifically at the carboxyl side of the internal proline residue, but could not hydrolyze long-chain bovine insulin B invitro. Like the ligand-free structure, MaPOP bound to the transition-state analog inhibitor ZPR was also in a closed state, which was not modulated by the common `latching loop' found in other POPs. The substrate-assisted catalytic mechanism of MaPOP reported here may represent a common mechanism for all POPs. These results may facilitate a better understanding of the catalytic behavior of POPs under physiological conditions.

Structural and biochemical analyses of β-N-acetylhexosaminidase Am0868 from Akkermansia muciniphila involved in mucin degradation

β-N-acetylhexosaminidases from the gut microbes are found to be capable of cleaving the specific glycoside linkages in the process of mucin degradation that has relevance for human health. However, features of the enzyme used in regulating the sugar-degrading capacities of Akkermansia muciniphila have not been well defined. Here we reported the crystal structure of a novel β-N-acetylhexosaminidase from Akkermansia muciniphila (Am0868), which displayed a typical (β/α) 8 barrel fold with a GlcNAc bound to the active center. Crystallographic and subsequent mutagenic analyses confirmed that Asp326 and Glu327 are the key catalytic residues of Am0868. Furthermore, Am0868 exhibited high specificity to β-GlcNAc supporting the substrate-assisted catalytic mechanism. Am0868 was also active in a broad pH and temperature range but inhibited strongly by metal ions Zn2+ and Cu2+. Collectively, these results indicate that Am0868 has the potential for mucin hydrolysis under some severe conditions, which highlight the superiority of A. muciniphila surviving in gut.

A proposal for a substrate-assisted catalytic mechanism for serine peptidases

A proposal for a substrate-assisted catalytic mechanism for serine peptidases

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

The catalytic activity of some classes of natural RNA, named as ribozymes, has been discovered just in the past decades. In this paper, the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and in a twister ribozyme from Oryza sativa. The free energy profiles associated with a baseline substrate-assisted mechanism for the reaction in the enzyme and in solution were computed by means of free energy perturbation methods within hybrid QM/MM potentials, describing the chemical system by the M06-2× functional and the environment by means of the AMBER and TIP3P force fields. The results confirm that this is a stepwise mechanism kinetically controlled by the second step that involves the P–O5′ breaking bond concomitant with the proton transfer from the OP1 atom to the leaving O5′ atom. 18O kinetic isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid–base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P–O5′ bond cleavage.

Mechanism of Human Nucleocytoplasmic Hexosaminidase D.

Mammalian β-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these β-hexosaminidases, hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized β-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-order rate constant at pH 6.5–7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to improve our understanding of the physiological role played by this unusual enzyme.

The Details of Glycolipid Glycan Hydrolysis by the Structural Analysis of a Family 123 Glycoside Hydrolase from Clostridium perfringens

Clostridium perfringens is an opportunistic pathogen of humans and animals whose genome encodes a wide variety of putative carbohydrate-hydrolyzing enzymes that are increasingly being shown to be directed toward the cleavage of host glycans. Among these putative enzymes is a member of glycoside hydrolase family 123. Here we show that the recombinant enzyme (referred to as CpNga123) encoded by the gene cloned from C. perfringens strain ATCC 13124 (locus tag CPF_1473) is a β-N-acetylgalactosaminidase, similar to NgaP from Paenibacillus sp. TS12. Like NgaP, CpNga123 was able to cleave the terminal β-D-GalNAc-(1→4)-D-Gal and β-D-GalNAc-(1→3)-D-Gal motifs that would be found in glycosphigolipids. The X-ray crystal structure of CpNga123 revealed it to have an N-terminal β-sandwich domain and a (β/α)8-barrel catalytic domain with a C-terminal α-helical elaboration. The structures determined in complex with reaction products provide details of the −1 subsite architecture, catalytic residues, and a structural change in the active site that is likely required to enable hydrolysis of the glycosidic bond by promoting engagement of the substrate by the catalytic residues. The features of the active site support the likelihood of a substrate-assisted catalytic mechanism for this enzyme. The structures of an inactive mutant of CpNga123 in complex with intact GA2 and Gb4 glycosphingolipid motifs reveal insight into aglycon recognition and suggest that the kinked or pleated conformation of GA2 caused by the β-1,4-linkage between N-acetylgalactosamine and galactose, and the accommodation of this conformation by the enzyme active site, may be responsible for greater activity on GA2.

Activation of the APC/C Ubiquitin Ligase by Enhanced E2 Efficiency

The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates the final events of mitosis by catalyzing the ubiquitination and proteasomal destruction of securin, cyclins, and other substrates [1, 2]. Like other members of the RING family of E3s [3, 4], the APC/C catalyzes direct ubiquitin transfer from an E2-ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Activator might also stimulate catalytic activity [5, 6], but the underlying mechanisms are not known. Here, we dissected activator function using an artificial fusion substrate in which the N-terminal region of securin was linked to an APC/C core subunit. This fusion substrate bound tightly to the APC/C and was ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 670-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor, but also enhances catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, full E2 stimulation required activator interaction with degron motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates.

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for β-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near (4)E, whereas that of LNB-thiazoline is in a (4)C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Substrate-Assisted Catalytic Mechanism of O-GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding O-linked N-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of O-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer's disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase O-GlcNAc transferase (uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme-substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11,326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S(N)2-like mechanism, in which a nucleophilic attack by O(Ser), facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1-O(Ser) = 1.92 Å and C1-O1 = 3.11 Å. The activation energy for this passage was estimated to be ~20 kcal mol(-1). These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the N-acetamino group of the donor participates in the catalytic mechanism.

High Resolution Crystal Structure of the Endo-N-Acetyl-β-D-Glucosaminidase Responsible for the Deglycosylation of Hypocrea jecorina Cellulases

Endo-N-acetyl-β-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-β-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified. Here, the expression, purification and the 1.3Å resolution structure of the ENGase (Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) are reported. Although the mature protein is C-terminally processed with removal of a 46 amino acid peptide, the protein has a complete (β/α)8 TIM-barrel topology. In the active site, the proton donor (E131) and the residue stabilizing the transition state (D129) in the substrate assisted catalysis mechanism are found in almost identical positions as in the bacterial GH18 ENGases: Endo H, Endo F1, Endo F3, and Endo BT. However, the loops defining the substrate-binding cleft vary greatly from the previously known ENGase structures, and the structures also differ in some of the α-helices forming the barrel. This could reflect the variation in substrate specificity between the five enzymes. This is the first three-dimensional structure of a eukaryotic endo-N-acetyl-β-D-glucosaminidase from glycoside hydrolase family 18. A glycosylation analysis of the cellulases secreted by a Hypocrea jecorina Endo T knock-out strain shows the in vivo function of the protein. A homology search and phylogenetic analysis show that the two known enzymes and their homologues form a large but separate cluster in subgroup B of the fungal chitinases. Therefore the future use of a uniform nomenclature is proposed.

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT.

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.

Engineering chitinases for the synthesis of chitin oligosaccharides: Catalytic amino acid mutations convert the GH-18 family glycoside hydrolases into transglycosylases

Abstract Two family GH-18 chitinases mutated on the key catalytic amino acids were evaluated as “glycosynthases” for the coupling of oxazoline activated donor to chitin oligosaccharide (COs) acceptors obtained from microbial fermentation. Bacillus circulans WL-12 chitinase A1 ( Bc ChiA1) and Trichoderma harzanium chitinase 42 ( Th Chit42) were individually mutated on the three conserved carboxylic acids, all suggested to have a role in catalysis: the general acid/base glutamate and the two aspartates, defined as the putative stabilizer and its assistant. The mutants D200A and D202A of Bc ChiA1, together with D170N and to a lesser extent D170A of Th Chit42 proved to be active for chitinbiose oxazoline polymerization, and also for coupling reaction between Gal(β1 → 4) chitinbiose oxazoline and chitinpentaose at neutral pH. These mutants have additionally retained the ability to catalyze transglycosylation reaction on natural COs, whereas their hydrolytic activity is abolished. Such mutants can be considered as chitin transglycosylases, verifying also the fact that the two conserved aspartic acids, the stabilizer and its assistant, are not a prerequisite for the formation of oxazolinium intermediate by acetamido anchimeric assistance, i.e. for the substrate-assisted catalysis mechanism of family GH-18 chitinases.

Quantum Mechanics/Molecular Mechanics Modeling of Substrate-Assisted Catalysis in Family 18 Chitinases: Conformational Changes and the Role of Asp142 in Catalysis in ChiB

Family 18 chitinases catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin. The mechanism has been proposed to involve the formation of an oxazolinium ion intermediate via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile (instead of an enzyme residue). Here, we have modeled the first step of the chitin hydrolysis catalyzed by Serratia marcescens chitinase B for the first time using a combined quantum mechanics/molecular mechanics approach. The calculated reaction barriers based on multiple snapshots are 15.8-19.8 kcal mol(-1) [B3LYP/6-31+G(d)//AM1-CHARMM22], in good agreement with the activation free energy of 16.1 kcal mol(-1) derived from experiment. The enzyme significantly stabilizes the oxazolinium intermediate. Two stable conformations ((4)C(1)-chair and B(3,O)-boat) of the oxazolinium ion intermediate in subsite -1 were unexpectedly observed. The transition state structure has significant oxacarbenium ion-like character. The glycosyl residue in subsite -1 was found to follow a complex conformational pathway during the reaction ((1,4)B → [(4)H(5)/(4)E](++) → (4)C(1) ↔ B(3,O)), indicating complex conformational behavior in glycoside hydrolases that utilize a substrate-assisted catalytic mechanism. The D142N mutant is found to follow the same wild-type-like mechanism: the calculated barriers for reaction in this mutant (16.0-21.1 kcal mol(-1)) are higher than in the wild type, in agreement with the experiment. Asp142 is found to be important in transition state and intermediate stabilization.

The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii

BackgroundThe interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane.ResultsIrga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRα). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism.ConclusionsThe catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families.

Computational evidence for the substrate-assisted catalytic mechanism of O-GlcNAcase. A DFT investigation

A DFT computational investigation of the catalytic mechanism of O-GlcNAcase shows the existence of a substrate-assisted reaction pathway similar to that proposed in the literature on the basis of experimental evidence: the carbonyl oxygen of the N-acyl group bonded at C2 of the substrate pyranose ring attacks the anomeric carbon affording a bicyclic oxazoline intermediate and causing the breaking of the glycosidic bond and the expulsion of the aglycon. This occurs in a single kinetic step where the transfer of a proton from Asp-243 (behaving as a general base) to the leaving group is simultaneous to the cycle formation and departure of the aglycon (an activation barrier E(a) of 16.5 kcal mol(-1)). Even if the other component of the catalytic dyad (Asp-242) is not actually involved in a proton transfer (as commonly suggested), it plays an important role in the catalysis through a complex network of hydrogen bonds that contribute to lower the activation barrier. The transition state of the process resembles an oxocarbenium ion (half chair conformation with an approximately planar sp(2) anomeric carbon). Following the lines of previous experiments aimed to demonstrate the existence of a substrate-assisted mechanism, it is found that the computed E(a) increases when the hydrogen atoms of the N-acetyl group are replaced with one, two and three F atoms and that a good linear correlation exists between the activation barrier E(a) and the σ* Taft electronic parameter of the fluoro-substituted N-acetyl groups.

Mechanistic insights into cognate substrate discrimination during proofreading in translation

Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "Double-Sieve Model." Strikingly using solution based direct binding studies, NMR-heteronuclear single quantum coherence (HSQC) and isothermal titration calorimetry experiments, with an editing domain of threonyl-tRNA synthetase, we show that the cognate substrate can gain access and bind to the editing pocket. High-resolution crystal structural analyses reveal that functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A strategically positioned "catalytic water" molecule is excluded to avoid hydrolysis of the cognate substrate using a "RNA mediated substrate-assisted catalysis mechanism" at the editing site. The mechanistic proof of the critical role of RNA in proofreading activity is a completely unique solution to the problem of cognate-noncognate selection mechanism.

Visualizing the Reaction Coordinate of an O-GlcNAc Hydrolase

N-Acetylglucosamine beta-O-linked to serine and threonine residues of nucleocytoplasmic proteins (O-GlcNAc) has been linked to neurodegeneration, cellular stress response, and transcriptional regulation. Removal of O-GlcNAc is catalyzed by O-GlcNAcase (OGA) using a substrate-assisted catalytic mechanism. Here we define the reaction coordinate using chemical approaches and directly observe both a Michaelis complex and the oxazoline intermediate.