Nanoscale engineering of gold particles in 18th century Böttger lusters and glazes.

SignificanceThe exploration of gold-based colorants in glass and glazes led Nobel Laureate Richard Zsigmondy to the study of colloids, and to the development, with Henry Siedentopf, of the earliest microscopes capable of resolving such small length scales. Zsigmondy's studies were preceded by alchemical investigations starting in the 17th century that yielded the gold-based Purple of Cassius, and experiments in the early 18th century resulting in an unusual purple iridescent porcelain overglaze, called Böttger luster, at the Meissen Manufactory. We discuss the first nano-scale characterization of Böttger luster, its successful replication, and propose an explanation for its optical properties based on the physics of scattering and interference of nanoparticle arrays.

Structure and Reaction Mechanism of YcjR, an Epimerase That Facilitates the Interconversion of d-Gulosides to d-Glucosides in Escherichia coli.

YcjR from Escherichia coli K-12 MG1655 catalyzes the manganese-dependent reversible epimerization of 3-keto-α-d-gulosides to the corresponding 3-keto-α-d-glucosides as a part of a proposed catabolic pathway for the transformation of d-gulosides to d-glucosides. The three-dimensional structure of the manganese-bound enzyme was determined by X-ray crystallography. The divalent manganese ion is coordinated to the enzyme by ligation to Glu-146, Asp-179, His-205, and Glu-240. When either of the two active site glutamate residues is mutated to glutamine, the enzyme loses all catalytic activity for the epimerization of α-methyl-3-keto-d-glucoside at C4. However, the E240Q mutant can catalyze hydrogen-deuterium exchange of the proton at C4 of α-methyl-3-keto-d-glucoside in solvent D2O. The E146Q mutant does not catalyze this exchange reaction. These results indicate that YcjR catalyzes the isomerization of 3-keto-d-glucosides via proton abstraction at C4 by Glu-146 to form a cis-enediolate intermediate that is subsequently protonated on the opposite face by Glu-240 to generate the corresponding 3-keto-d-guloside. This conclusion is supported by docking of the cis-enediolate intermediate into the active site of YcjR based on the known binding orientation of d-fructose and d-psicose in the active site of d-psicose-3-epimerase.

Manganese-Induced Substrate Promiscuity in the Reaction Catalyzed by Phosphoglutamine Cytidylyltransferase from Campylobacter jejuni.

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP/phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate, but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn2+, Cj1416 catalyzes the formation of 12 different reaction products using l-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, l-serine phosphate, or 3-phospho-d-glycerate as the nucleophile to displace pyrophosphate from CTP.

Cytidine Diphosphoramidate Kinase: An Enzyme Required for the Biosynthesis of the O-Methyl Phosphoramidate Modification in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of gastroenteritis, produces a capsular polysaccharide that is derivatized with a unique O-methyl phosphoramidate (MeOPN) modification. This modification contributes to serum resistance and invasion of epithelial cells. Previously, the first three biosynthetic steps for the formation of MeOPN were elucidated. The first step is catalyzed by a novel glutamine kinase (Cj1418), which catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of the amide nitrogen of l-glutamine. l-Glutamine phosphate is used by cytidine triphosphate (CTP):phosphoglutamine cytidylyltransferase (Cj1416) to displace pyrophosphate from CTP to generate cytidine diphosphate (CDP)-l-glutamine, which is then hydrolyzed by γ-glutamyl-CDP-amidate hydrolase (Cj1417) to form cytidine diphosphoramidate (CDP-NH2). Here, we show that Cj1415 catalyzes the ATP-dependent phosphorylation of CDP-NH2 to form 3'-phospho-cytidine-5'-diphosphoramidate. Cj1415 will also catalyze the phosphorylation of adenosine diphosphoramidate (ADP-NH2) and uridine diphosphoramidate (UDP-NH2) but at significantly reduced rates. It is proposed that Cj1415 be named cytidine diphosphoramidate kinase.

Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase.

Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated ( Enz-X-Pß-Pγ) and phosphorylated ( Enz-X-Pß) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within ß-[18O4]-ATP in support of the formation of the Enz-X-Pß-Pγintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [15N-amide]-l-glutamine in direct support of the Enz-X-Pßintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and ß-l-aspartyl hydroxamate, in addition to l-glutamine.

Biosynthesis of Nucleoside Diphosphoramidates in Campylobacter jejuni.

Campylobacter jejuni is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. C. jejuni produces a capsular polysaccharide (CPS) that contains a unique O-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of l-glutamine to form l-glutamine phosphate. In this investigation, the metabolic fate of l-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-l-glutamine to generate cytidine diphosphoramidate and l-glutamate. The structures of the two novel intermediates, CDP-l-glutamine and cytidine diphosphoramidate, were confirmed by 31P nuclear magnetic resonance spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP:phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase.

Discovery of a Glutamine Kinase Required for the Biosynthesis of the O-Methyl Phosphoramidate Modifications Found in the Capsular Polysaccharides of Campylobacter jejuni.

Bacterial capsular polysaccharides (CPS) are complex carbohydrate structures that play a role in the overall fitness of the organism. Campylobacter jejuni, known for being a major cause of bacterial gastroenteritis worldwide, produces a CPS with a unique O-methyl phosphoramidate (MeOPN) modification on specific sugar residues. The formation of P-N bonds in nature is relatively rare, and the pathway for the assembly of the phosphoramidate moiety in the CPS of C. jejuni is unknown. In this investigation we discovered that the initial transformation in the biosynthetic pathway for the MeOPN modification of the CPS involves the direct phosphorylation of the amide nitrogen of l-glutamine with ATP by the catalytic activity of Cj1418. The other two products are AMP and inorganic phosphate. The l-glutamine-phosphate product was characterized using 31P NMR spectroscopy and mass spectrometry. We suggest that this newly discovered enzyme be named l-glutamine kinase.

Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 Agents.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1'-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2; (Li et al. (2021) J. Med. Chem. 64, 11,267-11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (K i = 11-60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642-650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2-8 µM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3-0.6 µM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

Dinitrosyl iron complexes (DNICs) as inhibitors of the SARS-CoV-2 main protease.

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-ß-d-glucose tetraacetate) and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-ß-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 µM for TGTA-RRE and 33 ± 2 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.

Hydrogel-based Additive Manufacturing of Lithium Cobalt Oxide.

Three-dimensional (3D) multicomponent metal oxides with complex architectures could enable previously impossible energy storage devices, particularly lithium-ion battery (LIB) electrodes with fully controllable form factors. Existing additive manufacturing approaches for fabricating 3D multicomponent metal oxides rely on particle-based or organic-inorganic binders, which are limited in their resolution and chemical composition, respectively. In this work, aqueous metal salt solutions are used as metal precursors to circumvent these limitations, and provide a platform for 3D printing multicomponent metal oxides. As a proof-of-concept, architected lithium cobalt oxide (LCO) structures are fabricated by first synthesizing a homogenous lithium and cobalt nitrate aqueous photoresin, and then using it with digital light processing printing to obtain lithium and cobalt ion containing hydrogels. The 3D hydrogels are calcined to obtain micro-porous self-similar LCO architectures with a resolution of ~100µm. These free-standing, binder- and conductive additive-free LCO structures are integrated as cathodes into LIBs, and exhibit electrochemical capacity retention of 76% over 100 cycles at C/10. This facile approach to fabricating 3D LCO structures can be extended to other materials by tailoring the identity and stoichiometry of the metal salt solutions used, providing a versatile method for the fabrication of multicomponent metal oxides with complex 3D architectures.

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells.

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.