Peptide Epimerase Responsible for d-Amino Acid Introduction in Poly-γ-glutamic Acid Biosynthesis.

Poly-γ-glutamic acid (PGA) is a natural polymer of d- and/or l-glutamic acid (Glu) linked by isopeptide bonds. We recently showed that PGA synthetase, an enzyme complex composed of PgsB, PgsC, and PgsA, uses only l-Glu for polymerization, and d-Glu residues are introduced by peptide epimerization. However, it remains unclear which of the three enzymes is responsible for epimerization because in vitro functional characterization of the membrane-associated PgsBCA complex has never been successful. Here, we performed gene exchange experiments and showed that PgsA is responsible for the epimerization. Additionally, we identified a region in PgsA that modulates epimerization activity based on homology modeling from the recently solved structure of MslH, which showed 53% identity to PgsA. Our results suggested that d/l-ratios of the PGA product can be altered by introducing amino acid substitutions in this region, which will be useful for the production of PGA with controlled d/l-ratios.

Substrate Flexibilities of Norbelladine Synthase and Noroxomaritidine/Norcraugsodine Reductase for Hydroxylated and/or Methoxylated Aldehydes.

Amaryllidaceae alkaloids are structurally diverse natural products with a wide range biological properties, and based on the partial identification of the biosynthetic enzymes, norbelladine would be a common intermediate in the biosynthetic pathways. Previous studies suggested that norbelladine synthase (NBS) catalyzed the condensation reaction of 3,4-dihydroxybenzaldehyde and tyramine to form norcraugsodine, and subsequently, noroxomaritidine/norcraugsodine reductase (NR) catalyzed the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of norcraugsodine to generate norbelladine. However, recent studies have highlighted possible alternative Amaryllidaceae alkaloid biosynthetic pathways via the formation of isovanillin and vanillin from the 4-O- and 3-O-methylation reactions of 3,4-dihydroxybenzaldehyde, respectively. Herein, we focused on NpsNBS and NpsNR, which were initially identified from Narcissus pseudonarcissus, and explored their substrate recognition tolerance by performing condensation reactions of tyramine with various benzaldehyde derivatives, to shed light on the Amaryllidaceae alkaloid biosynthetic pathway from the viewpoint of the enzymatic properties. The assays revealed that both NpsNBS and NpsNR lacked the abilities to produce 4'-O- and 3'-O-methylnorbelladine from isovanillin and vanillin with tyramine, respectively. These observations thus suggested that Amaryllidaceae alkaloids are biosynthesized from norbelladine, formed through the condensation/reduction reaction of 3,4-dihydroxybenzaldehyde with tyramine.

Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a ß-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

A Small-Molecule Inhibitor of Factor Inhibiting HIF Binding to a Tyrosine-flip Pocket for the Treatment of Obesity.

In animals limiting oxygen upregulates hypoxia-inducible factor (HIF) promoting a metabolic shift towards glycolysis. Factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that regulates HIF function by reducing its interaction with histone acetyl transferases. HIF levels are negatively regulated by the HIF prolyl hydroxylases (PHDs), which like FIH, are 2-oxoglutarate(2OG) oxygenases. Genetic loss of FIH promotes both glycolysis and aerobic metabolism. FIH has multiple non-HIF substrates making it challenging to connect its biochemistry with physiology. A structure-mechanism guided approach identified a highly potent in vivo active FIH inhibitor, ZG-2291, binding of which promotes a conformational flip of a catalytically important tyrosine, enabling selective inhibition of FIH over other JmjC subfamily 2OG oxygenases. Consistent with genetic studies, ZG-2291 promotes thermogenesis and ameliorates symptoms of obesity and metabolic dysfunction in ob/ob mice. The results reveal ZG-2291 as a useful probe for the physiological functions of FIH and identify FIH inhibition as a promising strategy for obesity treatment.

Structural basis for binding of the renal carcinoma target hypoxia-inducible factor 2α to prolyl hydroxylase domain 2.

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.

Which modality is better to diagnose high-grade transformation in retroperitoneal liposarcoma? Comparison of computed tomography, positron emission tomography, and magnetic resonance imaging.

BACKGROUND: Survival in patients with retroperitoneal liposarcoma (RPLS) depends on the surgical management of the dedifferentiated foci. The present study investigated the diagnostic yield of contrast-enhanced CT, 18F-fluorodeoxyglucose positron emission tomography (PET), and diffusion-weighted MRI in terms of dedifferentiated foci within the RPLS. METHODS: Patients treated with primary or recurrent RPLS who underwent the above imaging between January 2010 and December 2021 were retrospectively reviewed. The diagnostic accuracy of the three modalities for histologic subtype of dedifferentiated liposarcoma (DDLS) and French Federation of Cancer Center (FNCLCC) grade 2/3 were compared using receiver operating characteristic curves and areas under the curves (AUCs). RESULTS: The cohort involved 32 patients with 53 tumors; 30 of which exhibited DDLS and 31 of which did FNCLCC grades 2/3. The optimal thresholds for predicting DDLS were mean CT value of 31 Hounsfield Unit (HU) (AUC = 0.880, 95% CI 0.775-0.984; p < 0.001), maximum standardized uptake value (SUVmax) of 2.9 (AUC = 0.865 95% CI 0.792-0.980; p < 0.001), while MRI failed to differentiate DDLS. The cutoff values for distinguishing FNCLCC grades 1 and 2/3 were a mean CT value of 24 HU (AUC = 0.858, 95% CI 0.731-0.985; p < 0.001) and SUVmax of 2.9 (AUC = 0.885, 95% CI 0.792-0.978; p < 0.001). MRI had no sufficient power to separate these grades. CONCLUSIONS: Contrast-enhanced CT and PET were useful for predicting DDLS and FNCLCC grade 2/3, while MRI was inferior to these two modalities.

Phenolic Derivatives with Anti-Acetylcholinesterase Inhibitory Activities from Galeola nudifolia in Vietnam.

A new phenolic derivative, galeomalate A (1), together with five known structurally related compounds (2-6), was isolated from the ethyl acetate extract of Galeola nudifolia collected in Vietnam. The structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-TOF-MS, and CD data, and chemical conversion of the sugar moiety. All isolated compounds possessed acetylcholinesterase (AChE) inhibitory activities in a dose-dependent manner. Among them, compounds 2 and 3 exhibited the first and second highest inhibitory activity on AChE with IC50 values of 122.13 and 125.49 µM, respectively. Compounds 1 and 4-6 inhibited the AChE activity by mixed modes of action comprising competitive and non-competitive modes, whereas 2 and 3 exerted their inhibitory activities in a competitive manner. Molecular docking analyses suggested that the phenyl-ß-D-glucopyranoside unit of 2 and 3 bound to the active site of AChE for the competitive inhibitory activities, while the mixed inhibitory activity of 4 was due to the two binding patterns in the active-site and the active-site entrance of AChE. Furthermore, the docking studies indicated that 1, 5, and 6 would inhibit AChE in a mixed inhibitory manner by adopting three distinct binding patterns of the additional phenyl-ß-D-glucopyranoside unit at the active-site entrance.

Fluorinated derivatives of pyridine-2,4-dicarboxylate are potent inhibitors of human 2-oxoglutarate dependent oxygenases.

2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets. Improving the selectivity profile of broad-spectrum 2OG oxygenase inhibitors may help enable the identification of selective inhibitors for use in functional assignment work. We report the synthesis of F- and CF3-substituted derivatives of the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylate (2,4-PDCA). Their inhibition selectivity profile against selected functionally distinct human 2OG oxygenases was determined using mass spectrometry-based assays. F-substituted 2,4-PDCA derivatives efficiently inhibit the 2OG oxygenases aspartate/asparagine-ß-hydroxylase (AspH) and the JmjC lysine-specific N ε-demethylase 4E (KDM4E); The F- and CF3-substituted 2,4-PDCA derivatives were all less efficient inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, except for the C5 F-substituted 2,4-PDCA derivative which inhibited AspH with a similar efficiency as 2,4-PDCA. Notably, the introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA results in a substantial increase in selectivity for AspH over KDM4E compared to 2,4-PDCA. Crystallographic studies inform on the structural basis of our observations, which exemplifies how a small change on a 2OG analogue can make a substantial difference in the potency of 2OG oxygenase inhibition.

Human Oxygenase Variants Employing a Single Protein FeII Ligand Are Catalytically Active.

Aspartate/asparagine-ß-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.

Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis.

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto.

Bacillus subtilis subsp. natto secretes the ComXnatto pheromone as a quorum-sensing pheromone to produce poly-γ-glutamate for biofilm formation. The amino-acid sequence of the pheromone is Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue is post-translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C-terminal end of the precursor peptide ComXnatto . Here, we report the functional analysis of ComQnatto , which catalyzes a unique farnesyl-transfer reaction. ComQnatto recognizes not only full-length ComXnatto but also N- and/or C-terminal truncated ComXnatto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQnatto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.

Two Distinct Substrate Binding Modes for the Normal and Reverse Prenylation of Hapalindoles by the Prenyltransferase AmbP3.

The cyanobacterial prenyltransferase AmbP3 catalyzes the reverse prenylation of the tetracyclic indole alkaloid hapalindole U at its C-2 position. Interestingly, AmbP3 also accepts hapalindole A, a halogenated C-10 epimer of hapalindole U, and catalyzes normal prenylation at its C-2 position. The comparison of the two ternary crystal structures, AmbP3-DMSPP/hapalindole U and AmbP3-DMSPP/hapalindole A, at 1.65-2.00 Šresolution revealed two distinct orientations for the substrate binding that define reverse or normal prenylation. The tolerance of the enzyme for these altered orientations is attributed to the hydrophobicity of the substrate binding pocket and the plasticity of the amino acids surrounding the allyl group of the prenyl donor. This is the first study to provide the intimate structural basis for the normal and reverse prenylations catalyzed by a single enzyme, and it offers novel insight into the engineered biosynthesis of prenylated natural products.

Molecular Insight into the Mg2+ -Dependent Allosteric Control of Indole Prenylation by Aromatic Prenyltransferase AmbP1.

AmbP1 is a cyanobacterial aromatic prenyltransferase and a dedicated synthase for (R)-3-geranyl-3-isocyanovinyl indolenine (2), the biogenetic precursor for hapalindole-type alkaloids. The regioselective geranylation of cis-indolyl vinyl isonitrile (1) by the standalone AmbP1 to give 2 has been shown to require a magnesium ion (Mg2+ ) to suppress the formation of cis-2-geranylindolyl vinyl isonitrile (3). Here, we report high-resolution crystal structures of AmbP1 in complex with 1 and geranyl S-thiodiphosphate (GSPP) in the presence and absence of a Mg2+ effector. The comparative study of these structures revealed a unique allosteric binding site for Mg2+ that modulates the conformation of 1 in the active site of AmbP1 for its selective geranylation. This work defines the structural basis for AmbP1 catalysis in the biogenesis of hapalindole-type alkaloids and provides the first atomic-level insight to the allosteric regulation of prenyltransferases.

[A Case Report on a Successful Resection after FOLFIRI plus Cetuximab Therapy for Unresectable Colorectal Cancer with Multiple Liver Metastases].

The patient was a 66-year-old woman with a history of right breast cancer 20 years prior. Her chief complaint was hematochezia, and she was diagnosed as having rectal cancer. She underwent laparoscopic high anterior resection. We made a diagnosis of moderately differentiated adenocarcinoma, type 2, 25×20 mm, pMP, pN0, Stage I, KRAS being wild-type. Multiple liver metastases were detected 6 months after the surgery. Tumor contacted with grison. The tumor was not completely resected as evidenced by the small liver remnant volume. Conversion therapy was administered, and the patient received 6 courses of FOLFIRI plus cetuximab therapy. Alopecia and grade 1 eruption were observed as adverse effects of the chemotherapy. The tumor size was reduced, and we resected the tumor by performing right lobectomy and partial hepatectomy. At 1 year 3 months after surgery, no recurrence was observed.

Discovery of Key Dioxygenases that Diverged the Paraherquonin and Acetoxydehydroaustin Pathways in Penicillium brasilianum.

Paraherquonin (1), a fungal meroterpenoid produced by Penicillium brasilianum NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of 1 (the prh cluster) and elucidated the pathway up to berkeleydione (2), which serves as the key intermediate for the biosynthesis of 1 as well as many other meroterpenoids. Interestingly, the nonheme iron and α-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford 2 from preaustinoid A1 (6), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, P. brasilianum MG11, which produces acetoxydehydroaustin instead of 1, was found to have a gene cluster nearly identical to the prh cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts 6 but produces preaustinoid A3 (17) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species.

Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont.

The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.

Stereospecific prenylation of tryptophan by a cyanobacterial post-translational modification enzyme.

Prenylation is a key post-translational reaction to increase the structural diversity and bioactivity of peptides and proteins. Until now, only one post-translational modification enzyme, ComQ, has been identified to mediate the prenylation of a tryptophan residue in ribosomally synthesized peptides. Here, we report the in vitro characterization of KgpF, a novel prenyltransferase which transfers dimethylallyl moieties to tryptophan residues during kawaguchipeptin A biosynthesis. The stereospecific prenylation by KgpF was determined by a combination of in vitro dimethylallylation of Fmoc-tryptophan by KgpF and chemical synthesis of dimethylallylated Fmoc-tryptophan diastereomers. KgpF modified the tryptophan derivative with a dimethylallyl group at the 3 position of its indole ring, resulting in the formation of a tricyclic structure with the same scaffold as prenylation by ComQ, but with the opposite stereochemistry.

Structure of lasso peptide epimerase MslH reveals metal-dependent acid/base catalytic mechanism.

The lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with D-tryptophan at the C-terminus, and is derived from the precursor peptide MslA. MslH, encoded in the MS-271 biosynthetic gene cluster (msl), catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, leading to epi-MslA. The detailed catalytic process, including the catalytic site and cofactors, has remained enigmatic. Herein, based on X-ray crystallographic studies in association with MslA core peptide analogues, we show that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The crystal structure analysis, followed by site-directed mutagenesis, docking simulation, and ICP-MS studies demonstrate that MslH employs acid/base chemistry to facilitate the reversible epimerization of the C-terminal Trp21 of MslA, by utilizing two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif with a Ca(II) ion via water molecules.

Detecting inattentiveness caused by mind-wandering during a driving task: A behavioral study.

This study aims to investigate whether behavioral variability and participants' self-ratings can be used to detect mind-wandering while driving and to examine their effects on braking performance during a driving task. We created a novel driving task and added a sustained attention response task (SART). We examined the effects of mind-wandering on braking performance and whether mind-wandering could be detected from SART response variability. The within-subjects results showed that self-reports of inattentiveness during driving correlated significantly with SART response variability. Multiple regression analysis with brake reaction time as the dependent variable revealed a significant relationship between self-reports of inattentiveness and mind-wandering. However, there were no other consistent linear associations between mind-wandering and SART response variability. Our results not only suggest that inattentiveness to driving caused by mind-wandering impairs braking performance but also emphasize the importance and difficulty of detecting this state from behavioral data alone.

Arginase inhibitory activities of guaiane sesquiterpenoids from Curcuma comosa rhizomes.

Arginases are bimanganese enzymes involved in many human illnesses, and thus are targets for disease treatments. The screening of traditional medicinal plants demonstrated that an ethanol extract of Curcuma comosa rhizomes showed significant human arginase I and II inhibitory activity, and further fractionation led to the isolation of three known guaiane sesquiterpenoids, alismoxide (1), 7α,10α-epoxyguaiane-4α,11-diol (2) and guaidiol (3). Tests of their inhibitory activities on human arginases I and II revealed that 1 exhibited selective and potent competitive inhibition for human arginase I (IC50 = 30.2 µM), whereas the other compounds lacked inhibitory activities against human arginases. To the best of our knowledge, this is the first demonstration of human arginase I inhibitory activity by a sesquiterpenoid. Thus, 1 is a primary and specific inhibitory molecule against human arginase I.