Cosubstrate Binding Research Articles

Abstract SY21-02: Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH

Abstract Ubiquinone or coenzyme Q (CoQ) is perhaps best known as a key component of the mitochondrial electron transport chain and as an endogenous antioxidant. However, CoQ has another critical function in cells, acting as an essential cosubstrate for a number of important metabolic enzymes that are associated with the inner mitochondrial membrane, including dihydroorotate dehydrogenase (DHODH), choline dehydrogenase (CHDH), proline dehydrogenase 1 and 2 (PRODH1, PRODH2), and glycerol-3-phosphate dehydrogenase 2 (GPD2). These FAD-containing mitochondrial enzymes are all hypothesized to utilize a two-step ‘ping-pong’ mechanism of catalysis. In this kinetic model, the baseline conformation of the enzyme has specific affinity for its first cosubstrate (dihydroorotate, choline, proline, glycerol-3-phosphate) but not its second cosubstrate (CoQ). Binding of the first cosubstrate to the enzyme results in oxidation of the cosubstrate and reduction of FAD to FADH2. This reduced ‘intermediate’ form of the enzyme releases the byproduct of the first reaction and then binds CoQ. Upon binding of CoQ, FADH2 is oxidized back to FAD and CoQ is reduced to CoQH2. The oxidized form of the enzyme has a low affinity for CoQH2 and CoQH2 is therefore released, restoring the enzyme to its baseline ‘empty’ conformation. Although there is considerable biochemical evidence to support this ‘double-displacement’ model of CoQ-dependent catalysis, it has been challenging to study the structures of these enzymes in their catalytically-active states due to the profound insolubility of CoQ and due to technical challenges associated with producing highly purified recombinant forms of membrane-associated proteins. Perhaps the most well-studied CoQ-dependent metabolic enzyme is DHODH. DHODH catalyzes the fourth and rate-limiting step in the de novo pyrimidine biosynthesis pathway, oxidizing dihydroorotate to orotate, which is then converted to the nucleotide precursor UMP. Due to the critical requirement of rapidly dividing cells for pyrimidines, DHODH has been proposed as a therapeutic target in cancer, and a number of highly potent and highly specific DHODH inhibitors have been developed, all of which act as competitive inhibitors of CoQ. By binding and stabilizing the protein, these inhibitors have made it possible to solve the x-ray crystal structure of DHODH. These structural studies have revealed that the inhibitors (and presumably CoQ) bind in a narrow amphipathic tunnel that spans from the membrane-embedded surface of the protein to its FAD-containing catalytic core. These structural studies have also found that DHO or orotate can bind DHODH when the CoQ tunnel is occupied by drug. Given that the ‘ping-pong’ model of catalysis is predicated on the assumption that cosubstrates and byproducts cannot simultaneously bind enzyme, it would appear that either the ping-pong model does not accurately reflect the catalytic mechanism of DHODH or, alternatively, that the structure of DHODH bound to drug is not an accurate representation of the structure of DHODH bound to CoQ. In order to gain a greater understanding of the structure of the DHODH-CoQ complex, we undertook to solve the x-ray crystal structure of DHODH in complex with decylubiquinone (dUb), a CoQ analog that can act as an electron acceptor for DHODH in vitro. These studies revealed that the x-ray crystal structure of DHODH in its catalytically-active cofactor-bound state deviates in subtle but important ways from the known structure of drug-bound DHODH, providing novel insights into the catalytic mechanism of CoQ-dependent enzymes. Citation Format: Julie-Aurore Losman. Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY21-02.

Observing How Glutathione and S-Hexyl Glutathione Bind to Glutathione S-Transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

Ethylene is an important signaling molecule in plants that triggers the growth of leaves, flowers, and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate into three CO2 molecules and ethylene. As the detailed mechanism of EFE in the biosynthesis of ethylene remains controversial and particularly the function of the co-substrate l-arginine, we decided to pursue a density functional theory study on the possible pathways of the enzyme leading to ethylene biosynthesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created, which contains all the features of the first- and second-coordination sphere of the active site and substrate (α-ketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either react with a molecule of CO2 to form a carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both the bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form a carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than the typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of the l-arginine co-substrate into the model leads to minor changes in the structure and fold, but its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, the key activation barriers seem less affected by the inclusion of l-arginine into the model. We, therefore, believe that the role of l-arginine is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO2. Our studies show that due to the distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO2 on a persuccinate complex to form carbonate products, leading to ethylene formation. This work gives suggestions on the engineering of EFE into a hydroxylase or improving the ethylene biosynthesis.

Optimizing the Substrate Uptake Rate of Solute Carriers.

The diversity in solute carriers arose from evolutionary pressure. Here, we surmised that the adaptive search for optimizing the rate of substrate translocation was also shaped by the ambient extracellular and intracellular concentrations of substrate and co-substrate(s). We explored possible solutions by employing kinetic models, which were based on analytical expressions of the substrate uptake rate, that is, as a function of the microscopic rate constants used to parameterize the transport cycle. We obtained the defining terms for five reaction schemes with identical transport stoichiometry (i.e., Na+: substrate = 2:1). We then utilized an optimization algorithm to find the set of numeric values for the microscopic rate constants, which provided the largest value for the substrate uptake rate: The same optimized rate was achieved by different sets of numerical values for the microscopic rate constants. An in-depth analysis of these sets provided the following insights: (i) In the presence of a low extracellular substrate concentration, a transporter can only cycle at a high rate, if it has low values for both, the Michaelis–Menten constant (KM) for substrate and the maximal substrate uptake rate (Vmax). (ii) The opposite is true for a transporter operating at high extracellular substrate concentrations. (iii) Random order of substrate and co-substrate binding is superior to sequential order, if a transporter is to maintain a high rate of substrate uptake in the presence of accumulating intracellular substrate. Our kinetic models provide a framework to understand how and why the transport cycles of closely related transporters differ.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

Catalytic Subunit of PKA as a Prototype of the Eukaryotic Protein Kinase Family.

The catalytic subunit of protein kinase A (PKAc) is conserved in all eukaryotic protein kinases. PKAc consists of two lobes that form the catalytic cleft containing the ATP-binding, peptide-binding site, and catalytic sites. During folding, PKAc secondary structures organize so that the non-polar regions form a globular core, while mobile loops and tails are exposed and can act as regulatory elements. De novo synthesized PKAc is phosphorylated at the T-loop, resulting in the formation of the active center capable of high-affinity binding of co-substrates. The ATP-molecule "sticks" the two lobes together, whereas the binding of peptide substrate completes the active center formation. The resulting catalytic triad (γ-phosphate of ATP, hydroxyl of Ser/Thr residue of the protein substrate, and Asp166 carboxyl) occupies a position optimal for catalysis. During the catalytic cycle, dynamic reorganization of polar and hydrophobic interactions ensures PKAc transition from the open to the closed conformation and vice versa. Understanding the structural basis of functioning of eukaryotic protein kinases (ePKs) is essential for successful design of ePK modulators.

Homology Modeling of Human Uridine-5'-diphosphate-glucuronosyltransferase 1A6 Reveals Insights into Factors Influencing Substrate and Cosubstrate Binding.

The elimination of numerous endogenous compounds and xenobiotics via glucuronidation by uridine-5′-diphosphate glycosyltransferase enzymes (UGTs) is an essential process of the body’s chemical defense system. UGTs have distinct but overlapping substrate preferences, but the molecular basis for their substrate specificity remains poorly understood. Three-dimensional protein structures can greatly enhance our understanding of the interactions between enzymes and their substrates, but because of the inherent difficulties in purifying and crystallizing integral endoplasmic reticulum membrane proteins, no complete mammalian UGT structure has yet been produced. To address this problem, we have created a homology model of UGT1A6 using I-TASSER to explore, in detail, the interactions of human UGT1A6 with its substrates. Ligands were docked into our model in the presence of the cosubstrate uridine-5′-diphosphate-glucuronic acid, interacting residues were examined, and poses were compared to those cocrystallized with various plant and bacterial glycosyltransferases (GTs). Our model structurally resembles other GTs, and docking experiments replicated many of the expected UGT-substrate interactions. Some bias toward the template structures’ protein–substrate interactions and binding preferences was evident.

Kinetic Models of Secondary Active Transporters.

Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding principle of kinetic modeling. We argue, however, that the level of acceptable parsimony is limited by the need to account for the substrate gradient established by a secondary active transporter, and for random order binding of co-substrates and substrate. Random order binding has consistently been observed in transporters of the SLC group.

Molecular regulation of lysine trimethylation by the P. aeruginosa methyltransferase EftM

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe life‐threatening lung and other infections in individuals with cystic fibrosis, compromised immune systems or severe burns. P. aeruginosa is also intrinsically resistant to many antibiotics making infections difficult to treat once established. We previously showed that the S‐adenosyl‐L‐methionine‐dependent methyltransferase EftM is responsible for trimethylation of lysine 5 of the translation factor EF‐Tu and that this modification enhances P. aeruginosa adhesion to host epithelial cells. Additionally, methyltransferase activity, and thus EF‐Tu modification, is temperature regulated via the intrinsic thermolability of EftM. Understanding EftM's complete mechanism of action may reveal new strategies for developing effective therapeutics targeting this pathogen. As the next step in this process, we now aim to define the substrate recognition and catalytic mechanisms of EftM. A phylogenetic reconstruction of the larger EftM family was generated to identify conservation patterns of putative key residues involved in interaction with EF‐Tu and facilitating catalysis. This analysis was supported by generation of an EftM homology model and protein‐protein docking studies to propose a EftM:EF‐Tu binding model. This model and phylogentic insights were used to guide site‐directed mutagenesis of residues in EftM and EF‐Tu to test their role in protein‐protein recognition using methyltransferase activity and binding assays. Finally, to begin investigating the role of protein dynamics and potential allosteric effects of SAM cosubstrate binding, other computational methods such as normal mode analysis were used to propose a coarse‐grain model of dynamics of EftM in parallel with experimental hydrogen‐deuterium exchange coupled with mass spectrometry (HDX‐MS) analysis of EftM and its complexes with SAM and EF‐Tu. In conclusion, our results provide the first structural insight into the EftM:EF‐Tu recognition, which paves the way for further understanding EftM's substrate recognition and catalytic mechanisms.Support or Funding InformationCystic Fibrosis Foundation Postdoctoral Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural basis for activation of SAGA histone acetyltransferase Gcn5 by partner subunit Ada2

The Gcn5 histone acetyltransferase (HAT) subunit of the SAGA transcriptional coactivator complex catalyzes acetylation of histone H3 and H2B N-terminal tails, posttranslational modifications associated with gene activation. Binding of the SAGA subunit partner Ada2 to Gcn5 activates Gcn5's intrinsically weak HAT activity on histone proteins, but the mechanism for this activation by the Ada2 SANT domain has remained elusive. We have employed Fab antibody fragments as crystallization chaperones to determine crystal structures of a yeast Ada2/Gcn5 complex. Our structural and biochemical results indicate that the Ada2 SANT domain does not activate Gcn5's activity by directly affecting histone peptide binding as previously proposed. Instead, the Ada2 SANT domain enhances Gcn5 binding of the enzymatic cosubstrate acetyl-CoA. This finding suggests a mechanism for regulating chromatin modification enzyme activity: controlling binding of the modification cosubstrate instead of the histone substrate.

Structural and functional characterization of protein\u2013lipid interactions of the Salmonella typhimurium melibiose transporter MelB

BackgroundMembrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein.ResultsLipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy.Conclusions(1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.

Inhibition of histone lysine methyltransferases G9a and GLP by ejection of structural Zn(II)

Histone lysine methyltransferases G9a and GLP are validated targets for the development of new epigenetic drugs. Most, if not all, inhibitors of G9a and GLP target the histone substrate binding site or/and the S-adenosylmethionine cosubstrate binding site. Here, we report an alternative approach for inhibiting the methyltransferase activity of G9a and GLP. For proper folding and enzymatic activity, G9a and GLP contain structural zinc fingers, one of them being adjacent to the S-adenosylmethionine binding site. Our work demonstrates that targeting these labile zinc fingers with electrophilic small molecules results in ejection of structural zinc ions, and consequently inhibition of the methyltransferase activity. Very effective Zn(II) ejection and inhibition of G9a and GLP was observed with clinically used ebselen, disulfiram and cisplatin.

Identification of Key Residues for Enzymatic Carboxylate Reduction.

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

Thermodynamic cooperativity of cosubstrate binding and cation selectivity of Salmonella typhimurium MelB.

The Na+-coupled melibiose symporter MelB, which can also be coupled to H+ or Li+ transport, is a prototype for the glycoside-pentoside-hexuronide:cation symporter family. Although the 3-D x-ray crystal structure of Salmonella typhimurium MelB (MelBSt) has been determined, the symport mechanisms for the obligatory coupled transport are not well understood. Here, we apply isothermal titration calorimetry to determine the energetics of Na+ and melibiose binding to MelBSt, as well as protonation of this transporter. Studies of the thermodynamic cycle for the formation of the Na+-MelBSt-melibiose ternary complex at pH 7.45 reveal that the binding of Na+ and melibiose is cooperative. The binding affinity for one substrate (Na+ or melibiose) is increased by the presence of the other by about eightfold. The coupling free energies (ΔΔG) of either substrate binding are ∼5 kJ/mol, and binding of both substrates releases a free energy of ∼35 kJ/mol. Measurements of the Na+-binding enthalpy at three different pH values, including the pKa value of MelB, indicate that the binding of one Na+ displaces one H+ per MelBSt molecule. In addition, the absolute dissociation constants for Na+ and H+, determined by competitive binding, show that MelBSt is selective for H+ over Na+ by ∼1,000-fold at a pKa of 6.25. Thus, the Na+ coupling in MelBSt is based not on ion selectivity but on ion concentrations and competitive binding because of a much higher Na+ concentration under physiological conditions. Such a selectivity feature seems to be common for membrane transport proteins that can bind both H+ and Na+ at a common site.

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding.

The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of ALKBH7, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7’s ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.

Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes

Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins.

A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis

Capreomycin is a potent ribosome-targeting antibiotic that is an essential component of current antituberculosis treatments, particularly in the case of multidrug-resistant Mycobacterium tuberculosis (Mtb). Optimal capreomycin binding and Mtb ribosome inhibition requires ribosomal RNA methylation in both ribosome subunits by TlyA (Rv1694), an enzyme with dual 2'-O-methytransferase and putative hemolytic activities. Despite the important role of TlyA in capreomycin sensitivity and identification of inactivating mutations in the corresponding Mtb gene tlyA, which cause resistance to capreomycin, our current structural and mechanistic understanding of TlyA action remains limited. Here, we present structural and functional analyses of Mtb TlyA interaction with its obligatory co-substrate for methyltransferase activity, S-adenosyl-l-methionine (SAM). Despite adopting a complete class I methyltransferase fold containing conserved SAM-binding and catalytic motifs, the isolated TlyA carboxyl-terminal domain exhibits no detectable affinity for SAM. Further analyses identify a tetrapeptide motif (RXWV) in the TlyA interdomain linker as indispensable for co-substrate binding. Our results also suggest that structural plasticity of the RXWV motif could contribute to TlyA domain interactions, as well as specific recognition of its two structurally distinct ribosomal RNA targets. Our findings thus reveal a novel motif requirement for SAM binding by TlyA and set the stage for future mechanistic studies of TlyA substrate recognition and modification that underpin Mtb sensitivity to capreomycin.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Influence of Potential on the Electrodeposition of Co on Au(111) by In Situ STM and Reflectivity Measurements

We study the influence of the deposition potential on the electrochemical growth of Co on Au(111) in the thickness range 0–8 atomic layers (ML). In-situ STM observations evidence two growth regimes which depend on the deposition potential: at potentials <−1.2 VMSE, Co grows 2D with the initial formation of biatomic islands which rapidly cover the entire Au surface, followed by layer-by-layer growth; at potentials >−1.20 VMSE, Co grows 3D with the formation of large multilayer islands with flat top. In situ fast STM imaging of the Co dissolution process evidences a remarkable thickness dependence of the dissolution potential. Complementary electrochemical and in situ optical reflectivity characterizations of Co dissolution as a function of the Co thickness suggest that the equilibrium potential Co/Co2+ on Au is shifted by ∼−30 mV with respect to that measured on Co. This value is discussed in terms of Co-substrate binding energy.

Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

Uridine 5′-diphospho-glucuronosyltransferase-1A9 (UGT1A9) expressed in the liver, shows good sequence identity with UGT1A10, expressed in the intestine. Both uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms show comprehensive overlapping substrate selectivity but there are differences in stereoselectivity, regiospecificity and rate of glucuronidation of the substrates. Multiple sequence alignment analyses of UGT1A9 and UGT1A10 showed that 13% of the residues in N-terminal domain (NTD) are non-identical between them. Herein, authors attempted homology modelling of UGT1A9 and UGT1A10 and validation using software tools and reported mutagenic studies. A molecular docking study of the known substrates is performed on UGT1A9 and UGT1A10 homology models. The non-identical N-terminal residues ranging from 111 to 117 in UGT1A9 and UGT1A10 were identified to play a central role in the substrate selectivity. However, substrate binding is performed by Ser111, Gly115 and Leu117 in UGT1A10 and Gly111, Asp115 and Phe117 in UGT1A9. This study reports new residues in NTD, showing interaction with uridine 5′-diphospho-glucuronic acid which binds with C-terminal domain. Further, molecular dynamics simulations were carried out to study the role of non-identical residues in substrate identification. The study demonstrates the folding of the UGT enzyme, particularly, helix-loop-helix transition and movement of Nα3-2 helix, in response to substrate and co-substrate binding.