Structure‐Based Development of an Affinity Probe for Sirtuin 2

Sirtuins are NAD⁺‐dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ?‐amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2‐selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment‐based screening, and affinity pull‐down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.     

Design and Synthesis of New 9‐Substituted Norharmane Derivatives as Potential Sirt5 Inhibitors

Sirt5 is a potential new drug target for the treatment of cancer, Alzheimer's disease, and Parkinson's disease. Given that norharmane is an important chemical synthon for some biologically important compounds and 9‐substituted norharmane derivatives containing a negatively charged carboxyl group may accord with the characteristic of potential Sirt5 inhibitors, a series of novel 9‐substituted norharmane derivatives were synthesized. The chemical structures and purities of all the target compounds were characterized by ¹H NMR, ¹³C NMR, MS, and HPLC. By in vitro SIRT5 inhibitory assays, three compounds ( 1a , 3a , and 3b ) show over 30% inhibition ratios at concentration of 100 µM , and the most active compound 3b has 35% and 52% inhibition ratios at 30 µM and 100 µM , respectively. Docking analysis showed that compound 3b is likely to fit very well on the substrate binding site of Sirt5, and hence, we believe that compound 3b can serve as a lead compound for further efforts to develop specific Sirt5 inhibitors.     

Chain‐Terminating and Clickable NAD+ Analogues for Labeling the Target Proteins of ADP‐Ribosyltransferases

ADP‐ribosyltransferases (ARTs) use NAD⁺ as a substrate and play important roles in numerous biological processes, such as the DNA damage response and cell cycle regulation, by transferring multiple ADP‐ribose units onto target proteins to form poly(ADP‐ribose) (PAR) chains of variable sizes. Efforts to identify direct targets of PARylation, as well as the specific ADP‐ribose acceptor sites, must all tackle the complexity of PAR. Herein, we report new NAD⁺ analogues that are efficiently processed by wild‐type ARTs and lead to chain termination owing to a lack of the required hydroxy group, thereby significantly reducing the complexity of the protein modification. Due to the presence of an alkyne group, these NAD⁺ analogues allow subsequent manipulations by click chemistry for labeling with dyes or affinity markers. This study provides insight into the substrate scope of ARTs and might pave the way for the further developments of chemical tools for investigating PAR metabolism.     

Characterization of xylitol dehydrogenase from debaryomyces hansenii

The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells ofDebaryomyces hansenii was partially purified in two Chromatographic steps, and characterization studies were carried out in order to inves tigate the role of the xylitol dehydrogenase-catalyzed step in the regu lation of D-xylose metabolism. The enzyme was most active at pH 9.0–9.5, and exhibited a broad polyol specificity. The Michaelis con stants for xylitol and NAD⁺ were 16.5 and 0.55 mM, respectively. Ca²⁺, Mg²⁺, and Mn²⁺ did not affect the enzyme activity. Conversely, Zn²⁺, Cd²⁺, and Co²⁺ strongly inhibited the enzyme activity. It was concluded that NAD⁺-xylitol dehydrogenase from D.hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K_m value for xylitol, and therefore should be named L-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14). The reason D.hansenii is a good xylitol producer is not because of its value of K_m for xylitol, which is low enough to assure its fast oxidation by NAD⁺ xylitol dehydrogenase. However, a higher K_m value of xylitol dehydro genase for NAD⁺ compared to theK _m values of other xylose-ferment ing yeasts may be responsible for the higher xylitol yields.     

Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules

Sirtuins are protein deacylases regulating metabolism and stress responses, and are implicated in aging‐related diseases. Small molecule activators for the human sirtuins Sirt1‐7 are sought as chemical tools and potential therapeutics, such as for cancer. Activators are available for Sirt1 and exploit its unique N‐terminus, whereas drug‐like activators for Sirt2–7 are lacking. We synthesized and screened pyrrolo[1,2‐a]quinoxaline derivatives, yielding the first synthetic Sirt6 activators. Biochemical assays show direct, substrate‐independent compound binding to the Sirt6 catalytic core and potent activation of Sirt6‐dependent deacetylation of peptide substrates and complete nucleosomes. Crystal structures of Sirt6/activator complexes reveal that the compounds bind to a Sirt6‐specific acyl channel pocket and identify key interactions. Our results establish potent Sirt6 activation with small molecules and provide a structural basis for further development of Sirt6 activators as tools and therapeutics.     

Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold

Artificial multi‐enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH–MDH and G6pDH–LDH) through the control of NAD⁺ substrate channeling by specifically shifting NAD⁺ between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine.     

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH,or NAD+/10‐Oxogeranial: Reaction Mechanisms

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.     

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH,or NAD+/10‐Oxogeranial: Reaction Mechanisms

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10‐oxogeranial were solved. The 10‐oxogeranial substrate binds in a transoid‐O1‐C3 conformation and can be reduced by hydride addition to form the byproduct S‐10‐oxo‐citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis‐enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone‐5β‐reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10‐oxogeranial structure also serves as a model for β‐face hydride attack in progesterone 5β‐reductases and is of general interest in the context of asymmetric synthesis.     

Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide ( NAD⁺ ) has emerged as a crucial substrate for NAD⁺ -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^thAD⁺ , are described. Its preparation was accomplished by enzymatic conversion of synthetic ^th ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^thAD⁺ was reduced by alcohol dehydrogenase (ADH) to N^thADH and afforded ^thADP-ribose ( ^th ADPr ) upon hydrolysis by NAD⁺ -nucleosidase (NADase). Furthermore, N^thAD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^thADP-ribosyl)ation. Due to its high photophysical responsiveness, N^thAD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺ ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Employing Methylene Green Coated Carbon Nanotube Electrodes to Enhance NADH Electrocatalysis for Use in an Ethanol Biofuel Cell

We prepared and characterized electrocatalysts based on multiwalled carbon nanotubes (MWCNTs) coated with methylene green (MG). These electrocatalysts can regenerate nicotinamide adenine dinucleotide (NAD⁺), so they are potentially applicable in the field of bioelectronics. NADH oxidation occurs between 0.14±0.002 and 0.16±0.002 V vs. Ag/AgCl. The most efficient bioanode furnishes 88±7 µW cm^?2 and 500 µA cm^?2 and an open circuit voltage of 590±22 mV. In conclusion, we obtained a reliable and easy‐to‐prepare electrocatalyst that can regenerate NAD⁺ and may be applicable in biosensors and bioelectronic devices that use a wide range of NAD⁺‐dependent enzymes.     

Binding mode of inhibitors and Cryptosporidium parvum IMP dehydrogenase: A combined ligand- and receptor-based study

A combined ligand- and target-based approach was used to analyse the interaction models of Cryptosporidium parvum inosine 5’-monophosphate dehydrogenase (CpIMPDH) with selective inhibitors. First, a ligand-based pharmacophore model was generated from 20 NAD⁺ competitive CpIMPDH inhibitors with the HipHop module. The characteristic of the NAD⁺ binding site of CpIMPDH was then described, and the binding modes of the representative inhibitors were studied by molecular docking. The combination of the pharmacophore model and the docking results allowed us to evaluate the pharmacophore features and structural information of the NAD⁺ binding site of CpIMPDH. This research supports the proposal of an interaction model inside the NAD⁺ binding site of CpIMPDH, consisting of four key interaction points: two hydrophobic-aromatic groups, a hydrophobic-aliphatic group and a hydrogen bond donor. This study also provides guidance for the design of more potent CpIMPDH inhibitors for the treatment of Cryptosporidium infections.     

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7**

The sirtuins are NAD⁺-dependent lysine deacylases, comprising seven isoforms (SIRT1–7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.     

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

NAD+在多壁碳纳米管修饰电极上电化学催化还原的研究

The cyclic voltammetric (CV) behaviors of NAD^ were studied with a multi-walled carbon nanotubes (MWNTs) modified glassy carbon (GC) electrode. In 0.05 mol/L tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer solution (pH=6.9), the MWNTs modified electrode showed high electrocatalytic activity toward reduction of NAD^ .The electroreduction of NAD^ was an irreversible diffusion controlled process. The cathodic peak current increased linearly with increasing the concentration of NAD^ . The influences of scan rate, temperature and concentration were also investigated.     

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD⁺), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD⁺, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD⁺, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD⁺ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD⁺, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

Biological Nicotinamide Cofactor as a Redox‐Active Motif for Reversible Electrochemical Energy Storage

Nicotinamide adenine dinucleotide (NAD⁺) is one of the most well‐known redox cofactors carrying electrons. Now, it is reported that the intrinsically charged NAD⁺ motif can serve as an active electrode in electrochemical lithium cells. By anchoring the NAD⁺ motif by the anion incorporation, redox activity of the NAD⁺ is successfully implemented in conventional batteries, exhibiting the average voltage of 2.3 V. The operating voltage and capacity are tunable by altering the anchoring anion species without modifying the redox center itself. This work not only demonstrates the redox capability of NAD⁺, but also suggests that anchoring the charged molecules with anion incorporation is a viable new approach to exploit various charged biological cofactors in rechargeable battery systems.     

NAD<Superscript>+</Superscript> metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity

Background

NAD⁺ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD⁺-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD⁺ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD⁺ to nicotinamide ratio and the NAD⁺ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD⁺ metabolism requires analytical definition and quantification of the key metabolites.

Results

Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD⁺ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD⁺ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD⁺ or nicotinamide levels in ways that would increase Sir2 activity.

Conclusions

The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

     

A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer‐membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1′) and nearest‐neighbor positions (P2, P2′) and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400‐fold improvement in OmpT catalytic efficiency, with a k _cat/K _m value of 6.1×10⁶ L mol⁻¹ s⁻¹. Wild‐type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.     

Effect of Enzyme and Cofactor Immobilization on the Response of Ethanol Oxidation in Zirconium Phosphate Modified Biosensors

Two different self‐contained ethanol amperometric biosensors incorporating layered [Ru(phend)₂bpy]²⁺‐intercalated zirconium phosphate (ZrP) as the mediator as well as yeast‐alcohol dehydrogenase (y‐ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) were constructed to improve upon a design previously reported where only this mediator was immobilized in the surface of a modified electrode. In the first biosensor, a [Ru(phend)₂bpy]²⁺‐intercalated ZrP modified carbon paste electrode (CPE) was improved by immobilizing in its surface both y‐ADH and NAD⁺ using quaternized Nafion membrane. In the second biosensor, a glassy carbon electrode was modified with [Ru(phend)₂bpy]²⁺‐intercalated ZrP, y‐ADH, and NAD⁺ using Nafion as the holding matrix. Calibration plots for ethanol sensing were constructed in the presence and absence of ZrP. In the absence of ZrP in the surface of the modified glassy carbon electrode, leaching of ADH was observed as detected by UV‐vis spectrophotometry. Ethanol sensing was also tested in the presence and absence of ascorbate to measure the selectivity of the sensor for ethanol. These two ethanol biosensors were compared to a previously reported one where the y‐ADH and the NAD⁺ were in solution, not immobilized.