Active Site Lysine Research Articles

Sirtuin-dependent reversible lysine acetylation of the o-succinylbenzoyl-coenzyme A synthetase of Bacillus subtilis.

Reversible lysine acylation (RLA) is a conserved posttranslational modification that cells of all domains of life use to regulate the biological function of proteins, some of which have enzymatic activity. Many AMP-forming organic acid:CoA ligases are regulated via acylation in prokaryotes and eukaryotes. Here, we report the acetylation of the o-succinylbenzoyl-CoA synthetase (EC 6.2.1.26) of Bacillus subtilis (BsMenE) by the GCN5-related acetyltransferase (GNAT) AcuA enzyme of this bacterium. BsMenE is part of the metabolic pathway that assembles menaquinone (MK), an essential component of the electron transport chain in B. subtilis. We demonstrate that the active-site lysine 471 (K471) of BsMenE is acetylated specifically by BsAcuA, and that acetylated BsMenE (BsMenEAc) is deacetylated by the NAD+-dependent sirtuin (BsSrtN) of this bacterium. The in vivo analyses performed in this study were done in an Escherichia coli ΔmenE strain because the enzymatic activity of MenE is essential in B. subtilis, but not in E. coli. The use of a heterologous system allowed us to assess the effect of acetylation on BsMenE function under MK-dependent growth conditions. Based on our in vivo data, we suggest that regulation of BsMenE by RLA reduces MK production, negatively affecting the growth rate and yield of the culture.IMPORTANCEReversible lysine acylation (RLA) is a posttranslational modification used by all cells to rapidly control the biological function of proteins. Herein, we identify an acetyltransferase and deacetylase in the soil bacterium Bacillus subtilis that can modify/demodify an enzyme required for the synthesis of menaquinone (MK), an essential electron carrier involved in respiration in cells of all domains of life. Based on our data, we suggest that under some as-yet-undefined physiological conditions, B. subtilis modulates MK biosynthesis, which changes the flux of electrons through the electron transport chain of this bacterium. To our knowledge, this is the first example of control of respiration by RLA.

Bacterial Phosphorylation Suppresses Carbapenemase Activity of the Class-D β-Lactamase OXA-24/40 from Acinetobacter baumannii.

The resistance of Gram-negative bacteria to β-lactam antibiotics is mostly due to deactivation of the antibiotics by bacterial enzymes, β-lactamases. Disclosing the factors regulating β-lactamase activity is vital for developing therapies to combat multidrug-resistant pathogens, such as Acinetobacter baumannii. Recent A. baumannii studies have revealed post-translational phosphorylation of serine β-lactamases at the active site serine. However, the functional consequences of such phosphorylation are unclear. We have taken the first steps to define these consequences through studies of OXA-24/40, a carbapenem-hydrolyzing class D β-lactamase in A. baumannii. We generated OXA-24/40 phosphorylated at its active site serine, S81, and explored its effects via NMR and MS. Phosphorylation inhibits carbapenemase activity by altering the active site conformation and impeding the carboxylation of an active site lysine, a requirement for class D β-lactamase activity. The inhibition varies with the carbapenem side chain properties. Phosphorylation-induced chemical shift perturbations extend beyond the active site, suggesting allosteric effects. Our findings offer the first atomic-level insights into the functional consequences of serine phosphorylation of class D β-lactamases.

Regulation of Absorption and Emission in a Protein/Fluorophore Complex.

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

The role of the active site lysine residue on FAD reduction by NADPH in glutathione reductase

Glutathione reductase (GR) is a two dinucleotide binding domain flavoprotein (tDBDF) that catalyzes the reduction of glutathione disulfide to glutathione coupled to the oxidation of NADPH to NADP+. An interesting feature of GR and other tDBDFs is the presence of a lysine residue (Lys-66 in human GR) at the active site, which interacts with the flavin group, but has an unknown function. To better understand the role of this residue, the dynamics of GR was studied using molecular dynamics simulations, and the reaction mechanism of FAD reduction by NADPH was studied using QM/MM molecular modeling. The two possible protonation states of Lys-66 were considered: neutral and protonated. Molecular dynamics results suggest that the active site is more structured for neutral Lys-66 than for protonated Lys-66. QM/MM modeling results suggest that Lys-66 should be in its neutral state for a thermodynamically favorable reduction of FAD by NADPH. Since the reaction is unfavorable with protonated Lys-66, the reverse reaction (the reduction of NADP+ by FADH−) is expected to take place. A phylogenetic analysis of various tDBDFs was performed, finding that an active site lysine is present in different the tDBDFs enzymes, suggesting that it has a conserved biological role. Overall, these results suggest that the protonation state of the active site lysine determines the energetics of the reaction, controlling its reversibility.

Structure of the Oxygen, Pyridoxal Phosphate-Dependent Capuramycin Biosynthetic Protein Cap15.

Pyridoxal phosphate-dependent enzymes able to use oxygen as a co-substrate have emerged in multiple protein families. Here, we use crystallography to solve the 2.40 Å resolution crystal structure of Cap15, a nucleoside biosynthetic enzyme that catalyzes the oxidative decarboxylation of glycyl uridine. Our structural study captures the internal aldimine, pinpointing the active site lysine as K230 and showing the site of phosphate binding. Our docking studies reveal how Cap15 is able to catalyze a stereoselective deprotonation reaction, and bioinformatic analysis reveals active site residues that distinguish Cap15 from the structurally related d-glucosaminate-6-phosphate ammonia lyase and l-seryl-tRNA(Sec) selenium transferase (SelA). Our work provides the structural basis for further mechanistic investigation of a unique biosynthetic enzyme and provides a blueprint for understanding how oxygen reactivity emerged in the SelA-like protein family.

Isoform-specific optical activation of kinase function reveals p38-ERK signaling crosstalk.

Evolution has diversified the mammalian proteome by the generation of protein isoforms that originate from identical genes, e.g., through alternative gene splicing or post-translational modifications, or very similar genes found in gene families. Protein isoforms can have either overlapping or unique functions and traditional chemical, biochemical, and genetic techniques are often limited in their ability to differentiate between isoforms due to their high similarity. This is particularly true in the context of highly dynamic cell signaling cascades, which often require acute spatiotemporal perturbation to assess mechanistic details. To that end, we describe a method for the selective perturbation of the individual protein isoforms of the mitogen-activated protein kinase (MAPK) p38. The genetic installation of a photocaging group at a conserved active site lysine enables the precise light-controlled initiation of kinase signaling, followed by investigation of downstream events. Through optical control, we have identified a novel point of crosstalk between two major signaling cascades: the p38/MAPK pathway and the extracellular signal-regulated kinase (ERK)/MAPK pathway. Specifically, using the photoactivated p38 isoforms, we have found the p38γ and p38δ variants to be positive regulators of the ERK signaling cascade, while confirming the p38α and p38β variants as negative regulators.

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2−) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase.

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ=+51/Ψ=-143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β-sheet dihedral values of Φ=-135/Ψ=+170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5'AMP.

Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120–130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5′AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5′AMP unusually slowly, relative to the two phosphorylation reactions.

Free Energy Landscape and Proton Transfer Pathways of the Transimination Reaction at the Active site of the Serine Hydroxymethyltransferase Enzyme in Aqueous Medium.

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme belonging to the fold type I or aspartate aminotransferase (AspAT) family of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. Like other PLP-dependent enzymes, SHMT also undergoes the so-called transimination reaction before exhibiting its enzymatic activity. The transimination process constitutes an important pre-step for all PLP-dependent enzymes, where an internal aldimine of the PLP-enzyme complex gets converted to an external aldimine of the substrate-PLP complex at the active site of the enzyme. In case of the transimination reaction involving SHMT, the PLP molecule bound to the active site lysine residue of SHMT (internal aldimine) gets detached from the enzyme by a serine substrate to produce an external aldimine complex, where the PLP is now bound to the serine substrate. In the current study, the free energy surfaces and reaction pathways of different steps of the transimination reaction at the active site of SHMT are investigated by employing hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with metadynamics methods of rare event sampling. It is found that the process of transimination involving serine and PLP at the active site of the SHMT enzyme takes place through different elementary steps such as the formation of the first geminal diamine intermediate (GDI1), transfer of a proton from the substrate serine to the phenolic oxygen of PLP, followed by another proton transfer from PLP to the amine nitrogen of lysine with the formation of the second geminal diamine intermediate (GDI2), and finally, detachment of the active site lysine residue from PLP to produce the external aldimine.

Sirtuin-Dependent Reversible Lysine Acetylation Controls the Activity of Acetyl Coenzyme A Synthetase in Campylobacter jejuni.

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group (Nε) of an active-site lysine of the AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, this active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N-acetyltransferases (GNATs). Acs activity is lost as a result of acetylation and is restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c, cj1715, and cj1050c loci, namely, the AMP-forming acetate-CoA ligase (CjAcs), a type IV GCN5-type lysine acetyltransferase (GNAT [CjLatA]), and a NAD+-dependent (class III) sirtuin deacylase (CjCobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of CjAcs. IMPORTANCE This work provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase (CjAcs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of CjAcs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

Computational mechanistic study of human liver glycerol 3‐phosphate dehydrogenase using ONIOM method

AbstractHuman liver glycerol 3‐phosphate dehydrogenase catalyzes transfer of a hydride anion from NADH to dihydroxyacetone phosphate (DHAP) forming L‐glycerol 3‐phosphate and NAD+ in a single step reaction. It is proposed that a hydride ion is transferred from NADH to the carbonyl carbon of DHAP through a general acid catalysis in which the carbonyl oxygen of DHAP is protonated by a nearby acidic residue. Based on the crystal structure of enzyme, it was suggested that one of the active site lysine residues (Lys120/Lys204) might act as general acid for the protonation of the carbonyl oxygen at DHAP. In this study, we formulated a number of computational systems to study the hydride transfer mechanism including main active site amino acid side chains, NADH cofactor, and DHAP. The calculations involved ONIOM method consisting of DFT and molecular mechanics (MM). We evaluated the energetics of the hydride transfer process in different model systems while probing the roles of active site residues, Lys120/Lys204/Asp260. Based on calculations, protonated Asp260 has more favorable energetics to act as the general acid catalysis as compared to Lys120/204 residues.

Forbidden Protein Angles: Triosephosphate Isomerase as a Model for Studying Peripheral Residue Contribution to Catalysis.

Over the past 3.5 billion years of evolution, enzymes have adopted a seemingly infinite number of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be forbidden to all amino acids, save glycine, due to steric hindrance. Triosephosphate Isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site Lysine 13 (K13) with a dihedral within the forbidden zone (phi=69/psi=−139). Both the amino acid and the dihedral angle are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β‐sheet dihedral values of phi=−135/psi=170 but experiments show a 1,000‐fold loss in activity. Based on these results, we hypothesized that adopting the forbidden torsion angle for K13 contributes to catalysis. Using a combination of computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine from single to quadruple mutants. In Silico Molecular Dynamic (MD) simulations of 5×200ns per mutant species were made using 2JK2 unliganded Human TIM as a starting structure. Heat Maps, containing 400,000 K13 dihedral values per run reveal full or partial loss of forbidden zone angles depending on mutant combination. Initial experimental results with E97A single mutant showed a three‐fold decrease in kcat with little change in the KM value suggesting a modest catalytic role for this residue. For this mutant the heatmaps revealed the K13 angle spends less than 30% of its time in the forbidden zone compared to over 50% occupation in the wildtype suggesting the rate decrease may be due to the mutation affecting the positioning of K13. In addition to ongoing In Vivo characterization of the remaining mutants, double mutant cycles will be used to evaluate their energetic connections with K13. As modern protein design relies on naturally occurring protein folds, understanding how to recreate forbidden angles may enhance catalytic rates in unnatural enzymes and achieve chemical reactions not yet envisioned by nature nor organic chemistry.Support or Funding InformationWe would like to thank California State University Long Beach and the Carl E. Riley Endowed STEM scholarship for their generous funding.Heat map of where K13 dihedral occupies space on Ramachandran Plot over 200ns of MD simulation using Wild Type Human Triosephosphate Isomerase (RCSB:2JK2).Figure 1

New AMP-forming acid:CoA ligases from Streptomyces lividans, some of which are posttranslationally regulated by reversible lysine acetylation.

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of thenewly identified CoA ligases were under NAD+ -dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging.

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits "turn-on" fluorescence, due to a weakly fluorescent aldehyde that "lights up" with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.

Staphylococcus aureus modulates the activity of acetyl-Coenzyme A synthetase (Acs) by sirtuin-dependent reversible lysine acetylation.

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl-coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5-related N-acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type-IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcsAc was deacetylated (hence reactivated) by the NAD+ -dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.

Synthesis of ortho-formylphenylphosphonic acids as covalent probes of active site lysines

During the course of an investigation of targeted inhibition of DNA polymerase beta (pol β) lyase activity using small molecules, we observed the formation of an aldimine between (2-formyl)phenylphosphonic acid (2FPP) and butylamine under basic aqueous conditions; complete deprotonation of the phosphonate group was required to stabilize the imine product. Results of computational docking studies suggested that the reaction of Lys-72 on the lyase active site with an aldehyde group could be facilitated by a proximal phosphonate, not only because of the phosphonate’s ability to mimic phosphate interacting with the DNA binding site, but also because of its ability to shield the imine against hydrolysis. Novel pol β lyase inhibitors were thus prepared using a 2FPP analogue with an amine linker; P-C bond formation in synthesis of this intermediate was possible with an unprotected aldehyde using palladium-catalyzed, microwave-assisted Michaelis–Arbuzov chemistry. These compounds, and structurally related derivatives lacking the aldehyde or phosphonate, were evaluated in an assay for pol β to assess their potential for inhibition.

Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

The first example of a hydroxylammonium derivative that causes a specific covalent modification of the active-site lysine residue of an aldolase enzyme, which is a promising target for anti-bacterial drug discovery, is reported.

Highly Sensitive Lysine Deacetylase Assay Based on Acetylated Firefly Luciferase

Lysine deacetylases (KDACs) play important roles in many physiological processes and are implicated in many human diseases. Hence, the search for modulators of KDACs is very active, and reliable assays for monitoring their activity are key to success. Here, we describe a new KDAC assay based on Firefly luciferase harboring an acetylation on an essential active site lysine. We show that several KDACs can reverse this modification and hence activate luciferase. This new assay is extremely sensitive, reliable, and fast and can be performed in a continuous format. We used this assay to screen a small library of compounds and identified several novel effectors of SirT2 with low micromolar activity.