Facilitate Substrate Binding Research Articles

Peptide Activator Stabilizes DJ-1 Structure and Enhances Its Activity

DJ-1 is a vital enzyme involved in the maintenance of mitochondrial health, and its mutation has been associated with an increased risk of Parkinson’s disease (PD). Effective regulation of DJ-1 activity is essential for the well-being of mitochondria, and DJ-1 is thus a potential target for PD drug development. In this study, two peptides (15EEMETIIPVDVMRRA29 and 47SRDVVICPDA56) were utilized with the aim of enhancing the activity of DJ-1. The mechanisms underlying the activity enhancement by these two peptides were investigated using hydrogen/deuterium exchange mass spectrometry (HDXMS). The HDXMS results revealed distinct mechanisms. Peptide 1 obstructs the access of solvent to the dimer interface and stabilizes the α/β hydrolase structure, facilitating substrate binding to a stabilized active site. Conversely, peptide 2 induces a destabilization of the α/β hydrolase core, enhancing substrate accessibility and subsequently increasing DJ-1 activity. The binding of these two peptides optimizes the activity site within the dimeric structure. These findings offer valuable insights into the mechanisms underlying the activity enhancement of DJ-1 by the two peptides, potentially aiding the development of new drugs that can enhance the activity of DJ-1 and, consequently, advance PD treatment.

An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P.aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.

Metallo-β-lactamase SMB-1 evolves into a more efficient hydrolase under the selective pressure of meropenem

Metallo-β-lactamases (MβLs) are the primary mechanism of resistance to carbapenem antibiotics. To elucidate how MβLs have evolved with the introduction and use of antibiotics, the mutation and evolution of SMB-1 from Serratia marcescens were investigated in microbial evolution plates containing discontinuous meropenem (MEM) concentration gradients. The results revealed 2-point mutations, A242G and S257R; 1 double-site mutation, C240G/E258G; and 3 frameshift mutations, M5, M12, and M13, which are all missense mutations situated at the C-terminus. Compared with that of the wild-type (WT), the minimum inhibitory concentrations (MICs) of MEM for A242G, C240G/E258G, M5, M12, and M13 increased at least 120-fold, and that of S257R increased 8-fold. The catalytic efficiency kcat/Km increased by 365% and 647%, respectively. Concerning the structural changes, the structure at the active site changed from an ordered structure to an unordered conformation. Simultaneously, the flexibility of loop 1 was enhanced. These changes increased the volume of the active site cavity; thus, this was more conducive to exposing the Zn2+ site, facilitating substrate binding and conversion to products. In A242G, structural changes in Gly-242 can be transmitted to the active region via a network of interactions between the side chains of Gly-242 and the amino acid side chains near the active pocket. Together, these results pointed to the process of persistent drug tolerance and resistance, the SMB-1 enzyme evolved into a more exquisite structure with increased flexibility and stability, and stronger hydrolysis activity via genetic mutations and structural changes.

Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

Reactivity Tuning of Metal‐Free Artificial Photoenzymes through Binding Site Specific Bioconjugation

AbstractThe design and development of artificial metal‐free photoenzymes aims to combine the selectivity of enzymatic reactions with the benefits of modern synthetic photocatalysts. Removing the need for rare earth metals and allowing for milder reaction conditions, leading to a more sustainable catalytic system. Here, we present the design of a novel artificial photoenzyme by integrating an organophotocatalytic moiety based on a donor‐acceptor design into a steroid carrier protein (SCP‐2L). SCP‐2L possesses a hydrophobic tunnel facilitating substrate binding in aqueous media. The photocatalyst was site‐selectively bound to three SCP‐2L variants, possessing a non‐native cysteine residue strategically placed around the hydrophobic tunnel of the protein. The three modified photoenzymes were shown to be selective for the oxidation of organic sulfides giving up to 192 turnovers.

EZH2 W113C is a gain-of-function mutation in B-cell lymphoma enabling both PRC2 methyltransferase activation and tazemetostat resistance

Polycomb Repressive Complex2 (PRC2) suppresses gene transcription by methylating lysine 27 of histone H3 (H3K27) and plays critical roles in embryonic development. Among the core PRC2 subunits, EZH2 is the catalytic subunit and EED allosterically activates EZH2 upon binding trimethylated H3K27 (H3K27me3). Activating mutations on Y641, A677, and A687 within the enzymatic SET domain of EZH2 have been associated with enhanced H3K27me3 and tumorigenicity of many cancers including B-cell lymphoma and melanoma. To tackle the critical residues outside the EZH2 SET domain, we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel gain-of-function (GoF) mutation W113C, which increases H3K27me3 in vitro and in vivo and promotes CDKN2A silencing to a similar level as the EZH2 Y641F. Different from other GoF mutations, this mutation is located in the SAL motif at the EZH2 N-terminus, which stabilizes the SET domain and facilitates substrate binding. This may explain how the W113C mutation increases PRC2 activity. Tazemetostat is an FDA-approved EZH2 binding inhibitor for follicular lymphoma treatment. Intriguingly, the W113C mutation leads to tazemetostat-resistance in cell and in vivo in both H3K27 methylation and tumor proliferation. Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance, effectively decreases H3K27me3, and blocks tumor proliferation in cells expressing EZH2 W113C. As this mutation is originally identified from lymphoma samples, our results demonstrated its activating characteristic and the deleterious consequence, provide insights on PRC2 regulation, and support the continued exploration of treatment optimization for lymphoma patients.

Engineering of Hydroxymandelate Oxidase and Cascade Reactions for High-Yielding Conversion of Racemic Mandelic Acids to Phenylglyoxylic Acids and (R)- and (S)-Phenylglycines

An alcohol oxidase (AOx) for the (S)-enantioselective oxidation of 4-hydroxymandelic acid (4-HMA) to 4-hydroxyphenylglyoxylic acid (4-HPGA) with enhanced activity was developed by directed evolution of hydroxymandelate oxidase (HMO) through three rounds of iterative saturation mutagenesis. The engineered HMO mutant A80G-T159S-T162Q (HMOTM) catalyzed the oxidation of (S)-4-HMA to 4-HPGA with a 23-fold enhancement in catalytic efficiency (kcat/KM). Substrate docking simulation on HMOTM suggested that A80G reoriented FMN, while T159S and T162Q formed hydrogen bonds with the carboxylic group of the substrate, thus facilitating substrate binding and catalysis. (S)-Enantioselective HMOTM was used together with mandelic acid racemase (MR) and catalase (KatE) to achieve high-yielding oxidation of rac-4-HMA to 4-HPGA with either purified enzymes (up to 93% yield and 426 mM) or Escherichia coli (HMC) cells expressing the three enzymes (up to 93% yield and 140 mM). The HMOTM-MR-KatE cascades were applied for the oxidation of seven other substituted MAs, producing the corresponding phenylglyoxylic acids with 90–99% conversions using purified enzymes or whole cells. Efficient conversion of racemic α-hydroxy acids to (S)- or (R)-α-amino acids was achieved by combining HMOTM-MR-KatE with (S)-enantioselective transaminase (EcαTA) or (R)-enantioselective transaminase (DpgAT), together with glutamate dehydrogenase (GluDH). Coupling of E. coli (HMC) with E. coli (E-G) expressing EcαTA and GluDH for one-pot biotransformation of five rac-MAs produced the corresponding (S)-phenylglycines (PGs) in 91–99% ee with 73–98% conversions. Using E. coli (HMC) and E. coli (D-G) expressing DpgAT and GluDH enabled the production of five (R)-PGs with 93–99% ee and 73–98% conversions from the corresponding rac-MAs. The engineered AOx, AOx-MR-KatE cascades, and AOx-MR-Kat-GluDH-(S)- or (R)-TA cascades provide useful tools for highly active and enantioselective oxidation of racemic hydroxyacids and high-yielding conversion of racemic hydroxyacids to ketoacids and enantiopure (S)- or (R)-aminoacids, respectively, which are challenging and useful chemical reactions.

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling.

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C-N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS.

Dynamic interactions in the l-lactate oxidase active site facilitate substrate binding at pH4.5

The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

PI3K Driver Mutations: A Biophysical Membrane-Centric Perspective.

Ras activates its effectors at the membrane. Active PI3Kα and its associated kinases/phosphatases assemble at membrane regions enriched in signaling lipids. In contrast, the Raf kinase domain extends into the cytoplasm and its assembly is away from the crowded membrane surface. Our structural membrane-centric outlook underscores the spatiotemporal principles of membrane and signaling lipids, which helps clarify PI3Kα activation. Here we focus on mechanisms of activation driven by PI3Kα driver mutations, spotlighting the PI3Kα double (multiple) activating mutations. Single mutations can be potent, but double mutations are stronger: their combination is specific, a single strong driver cannot fully activate PI3K, and two weak drivers may or may not do so. In contrast, two strong drivers may successfully activate PI3K, where one, for example, H1047R, modulates membrane interactions facilitating substrate binding at the active site (km) and the other, for example, E542K and E545K, reduces the transition state barrier (ka), releasing autoinhibition by nSH2. Although mostly unidentified, weak drivers are expected to be common, so we ask here how common double mutations are likely to be and why PI3Kα with double mutations responds effectively to inhibitors. We provide a structural view of hotspot and weak driver mutations in PI3Kα activation, explain their mechanisms, compare these with mechanisms of Raf activation, and point to targeting cell-specific, chromatin-accessible, and parallel (or redundant) pathways to thwart the expected emergence of drug resistance. Collectively, our biophysical outlook delineates activation and highlights the challenges of drug resistance.

Dimerization of PHGDH via the catalytic unit is essential for its enzymatic function

Human D-3-phosphoglycerate dehydrogenase (PHGDH), a key enzyme in de novo serine biosynthesis, is amplified in various cancers and serves as a potential target for anticancer drug development. To facilitate this process, more information is needed on the basic biochemistry of this enzyme. For example, PHGDH was found to form tetramers in solution and the structure of its catalytic unit (sPHGDH) was solved as a dimer. However, how the oligomeric states affect PHGDH enzyme activity remains elusive. We studied the dependence of PHGDH enzymatic activity on its oligomeric states. We found that sPHGDH forms a mixture of monomers and dimers in solution with a dimer dissociation constant of ∼0.58 μM, with the enzyme activity depending on the dimer content. We computationally identified hotspot residues at the sPHGDH dimer interface. Single-point mutants at these sites disrupt dimer formation and abolish enzyme activity. Molecular dynamics simulations showed that dimer formation facilitates substrate binding and maintains the correct conformation required for enzyme catalysis. We further showed that the full-length PHGDH exists as a dynamic mixture of monomers, dimers, and tetramers in solution with enzyme concentration-dependent activity. Mutations that can completely disrupt the sPHGDH dimer show different abilities to interrupt the full-length PHGDH tetramer. Among them, E108A and I121A can also disrupt the oligomeric structures of the full-length PHGDH and abolish its enzyme activity. Our study indicates that disrupting the oligomeric structure of PHGDH serves as a novel strategy for PHGDH drug design and the hotspot residues identified can guide the design process.

Inter domain interactions influence the substrate affinity and hydrolysis product specificity of xylanase from Streptomyces chartreusis L1105

PurposeThis study investigated the influence of inter-domain interactions on the substrate affinity and hydrolysis product specificity of xylanase.MethodsGenes encoding a GH10 endo-xylanase from Streptomyces chartreusis L1105 xynA and its truncated derivative were cloned and expressed in Escherichia coli. The catalytic activities of the enzyme (xynA) and the derivative xynADCBM, lacking the carbohydrate binding module (CBM), were assessed to evaluate the role of CBM in xynA.ResultsRecombinant xynA (44 kDa) was found to be optimally active on beechwood xylan at 65 °C with pH 7.7, while xynADCBM (34 kDa) exhibited optimal activity at 65 °C with pH 7.2. Additionally, xynA and xynADCBM were found to be highly thermostable at 40–60 °C, each retaining 80% of their original activity after 30 min. The xynADCBM without the CBM domain was highly efficient at hydrolyzing xylan to produce xylobiose (over 67%), which may be because the CBM domain facilitates substrate binding with xylanase. Meanwhile, the xylan hydrolysis efficiency of xynADCBM was higher than that of xynA.ConclusionThese findings showed that the CBM domain with non-catalytic activity has no significant effect on the characteristics of the enzyme at optimum pH and pH tolerance. It has also been suggested that the derivative xynADCBM without CBM components can promote hydrolysis of xylan to yield xylooligosaccharides, which has great potential economic benefits.

Structural Insight into the Mechanism of Staphylococcus aureus Stp1 Phosphatase.

Staphylococcus aureus Stp1, which belongs to the bacterial metal-dependent protein phosphatase (PPM) family, is a promising candidate for antivirulence targeting. How Stp1 recognizes the phosphorylated peptide remains unclear, however. In order to investigate the recognition mechanism of Stp1 in depth, we have determined a series of crystal structures of S.aureus Stp1 in different states and the structural complex of Stp1 bound with a phosphorylated peptide His12. Different phosphorylated peptides, including MgrA- and GraR-derived phosphopeptides, are substrates of Stp1, which supports the function of Stp1 as a selective Ser/Thr phosphatase. In addition, interestingly, the crystal structures of R161-Stp1 variants combined with the biochemical activity validations have uncovered that R161 residue plays a key role to control the conformation switches of the flap domain in order to facilitate substrate binding and the dephosphorylation process. Our findings provide crucial structural insight into the molecular mechanism of S.aureus Stp1 phosphatase and reveal the phosphorylated peptides for biochemistry study and inhibitor screening of Stp1.

Catechol Oxidase and SOD Mimicking by Copper(II) Complexes of Multihistidine Peptides

Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis–Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis–Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {Nim/2Nim,2N−} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range.

Divalent metal ions control activity and inhibition of protein kinases.

Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and orientation. Magnesium allows stable substrate binding and, on the other hand accelerates product release, thus being extremely efficient in turnover catalysis. We extended our studies to non-catalytic functions of protein kinases looking at pseudokinases, a subfamily of protein kinases inherently lacking critical residues for catalysis. Recently, pseudokinases have been linked to human diseases. Some pseudokinases are still capable of binding metal ions, yet have lost the ability to transfer the phosphoryl group from ATP to a given substrate. Here metal ions stabilize an active like, though catalytically unproductive conformation and are therefore crucial to maintain non-catalytic function. Finally, we demonstrate for the canonical kinase PKA that the trace metal manganese alone can stabilize protein kinases in an active like conformation allowing them to bind substrates even in the absence of nucleotides.

The "Lid" in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site.

Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a "lid", a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the "lid" in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.

Cap-domain closure enables diverse substrate recognition by the C2-type haloacid dehalogenase-like sugar phosphatase Plasmodium falciparum HAD1.

Haloacid dehalogenases (HADs) are a large enzyme superfamily of more than 500,000 members with roles in numerous metabolic pathways. Plasmodium falciparum HAD1 (PfHAD1) is a sugar phosphatase that regulates the methylerythritol phosphate (MEP) pathway for isoprenoid synthesis in malaria parasites. However, the structural determinants for diverse substrate recognition by HADs are unknown. Here, crystal structures were determined of PfHAD1 in complex with three sugar phosphates selected from a panel of diverse substrates that it utilizes. Cap-open and cap-closed conformations are observed, with cap closure facilitating substrate binding and ordering. These structural changes define the role of cap movement within the major subcategory of C2 HAD enzymes. The structures of an HAD bound to multiple substrates identifies binding and specificity-determining residues that define the structural basis for substrate recognition and catalysis within the HAD superfamily. While the substrate-binding region of the cap domain is flexible in the open conformations, this region becomes ordered and makes direct interactions with the substrate in the closed conformations. These studies further inform the structural and biochemical basis for catalysis within a large superfamily of HAD enzymes with diverse functions.

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE.

Telomerase catalyzes the addition of nucleotides to the ends of chromosomes to complete genomic DNA replication in eukaryotes and is implicated in multiple diseases, including most cancers. The core enzyme is composed of a reverse transcriptase and an RNA subunit, which provides the template for DNA synthesis. Despite extensive divergence at the sequence level, telomerase RNAs share several structural features within the catalytic core, suggesting a conserved enzyme mechanism. We have investigated the structure of the core of the human and yeast telomerase RNAs using SHAPE, which interrogates flexibility of each nucleotide. We present improved secondary-structure models, refined by addition of five base triples within the yeast pseudoknot and an alternate pairing within the human-specific element J2a.1 in the human pseudoknot, both of which have implications for thermodynamic stability. We also identified a potentially structured CCC region within the template that may facilitate substrate binding and enzyme mechanism. Overall, the SHAPE findings reveal multiple similarities between the Saccharomyces cerevisiae and Homo sapiens telomerase RNA cores.