The 5-ketofructose reductase of Gluconobacter sp. strain CHM43 is a novel class in the shikimate dehydrogenase family

Gluconobacter sp. CHM43 oxidizes mannitol to fructose and then does fructose to 5-keto-D-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP + -dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP + -dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the K M value for 5KF, but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the K M value for 5KF, suggesting a similar catalytic mechanism to that of SDH. Taken together, we suggest that KFR is a new member of the SDH family. Importance A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose at a high yield, a potential low calorie sweetener. Here we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small sub-group of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.

Two UGT84A Family Glycosyltransferases Regulate Phenol, Flavonoid, and Tannin Metabolism in Juglans regia (English Walnut)

We showed previously that gallic acid is produced in walnut from 3-dehydroshikimate by a shikimate dehydrogenase (JrSkDH). This study focuses on the next step in the hydrolysable tannin pathway, the formation of 1-O-galloyl-β-D-glucose from the phenolic gallic acid and UDP glucose by a glycosyltransferase. JrGGT1 (UGT84A73) and JrGGT2 (UGT84A74) are predicted to be two such glycosyltransferases, which we expressed in tobacco plants. GC-MS analysis of the transgenic tobacco revealed moderate, yet significant alterations in plant secondary metabolism, such as depleted phenolic acids, including gallic acid. We postulate that these effects are due to JrGGT1 and JrGGT2 activity, as JrGGT orthologs glycosylate these phenolic compounds in vitro. Moreover, JrGGT expression in tobacco caused upregulation of shikimic acid pathway metabolites and differing responses in phenylpropanoids, such as phenolic acids and flavonoids. In transcriptome analysis of walnut pellicle tissues, both JrGGTs showed substantial and significant expression correlations with the gallic acid-producing JrSkDHs and were highly coexpressed with the genetic circuits constituting the shikimic acid and phenylpropanoid biosynthetic pathways. Verification of JrGGT gene expression by transcriptome analysis of 20 walnut tissues revealed striking similarities with that of the pellicle data, with the greatest expression in roots, wood, buds, and leaves of Juglans regia cv. Chandler: tissues that typically accumulate hydrolysable tannins. Like the transgenic tobacco, pellicle metabolomic analyses revealed that many phenylpropanoids correlated negatively with JrGGT expression, while shikimic acid pathway metabolites correlated positively with JrGGT expression. This research supports the hypothesis that JrGGT1 and JrGGT2 play non-trivial roles in metabolism of phenolic acids, flavonoids, and ostensibly, tannins.

Dehydroquinate dehydratase/shikimate dehydrogenases involved in gallate biosynthesis of the aluminum-tolerant tree species Eucalyptus camaldulensis

Main conclusion Eucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B. Abstract The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Terpyridine-metal complexes: effects of different substituents on their physico-chemical properties and density functional theory studies

A series of different substituted terpyridine (tpy)-based ligands have been synthesized by Kröhnke method. Their binding behaviour was evaluated by complexing them with Co(II), Fe(II) and Zn(II) ions, which resulted in interesting coordination compounds with formulae, [Zn(tpy) 2 ]PF 6 , [Co(tpy) 2 ](PF 6 ) 2 , [Fe(tpy) 2 ](PF 6 ) 2 and interesting spectroscopic properties. Their absorption and emission behaviours in dilute solutions were investigated in order to explain structure–property associations and demonstrate the impact of different aryl substituents on the terpyridine scaffold as well as the role of the metal on the complexes. Photo-luminescence analysis of the complexes in acetonitrile solution revealed a transition from hypsochromic to bathochromic shift. All the compounds displayed remarkable photo-luminescent properties and various maximum emission peaks owing to the different nature of the functional groups. Furthermore, the anti-microbial potential of ligands and complexes was evaluated with docking analyses carried out to investigate the binding affinity of terpyridine-based ligands along with corresponding proteins (shikimate dehydrogenase and penicillin-binding protein) binding sites. To obtain further insight into molecular orbital distributions and spectroscopic properties, density functional theory calculations were performed for representative complexes. The photophysical activity and interactions between chromophore structure and properties were both investigated experimentally as well as theoretically.

Potency of Phytochemicals from Guava (Psidium guajava) Seeds against Escherichia coli to Cure Diarrhoea: An in silico Analysis

Machine learning to deep learning is the enduring upgrading in the pharmaceutical research field. Phytochemicals from Psidium guajava plant extract are traditionally used to cure Diarrhoea. The causative agent of the disease is Escherichia coli. To identify the secondary metabolites (ligand) which is the main responsive compound that have the capacity to inhibit the growth of microorganism was carried by molecular docking method “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that Heptadecanoic acid can effectively deactivate the shikimate dehydrogenase enzyme thereby interrupting the life cycle of the organism.

Moringa oleifera Derived Phytochemicals against Shikimate Dehydrogenase of Helicobacter pylori Causing Peptic Ulcer

Moringa oleifera plant extract is traditionally used to cure Peptic ulcer. It is caused by Helicobacter pylori. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that 9-octadecenoic acid can effectively deactivate the shikimate dehydrogenase enzyme thereby interrupting the life cycle of the organism.

Alkanna tinctoria Derived Phytochemicals against Staphylococcus aureus Causing Eczema

Alkanna tinctoria plant extract is traditionally used to heal eczema. It is caused by Staphylococcus aureus. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that 2-Hydroxy-3-Phenyl-1-4-naphthoquinone can can effectively deactivate the shikimate dehydrogenase enzyme thereby interrupting the life cycle of the organism.

Beet Root Derived Phytochemicals against Escherichia coli Causing Diarrhea

Phytochemicals from Beet Root (Beta vulgaris) plant extract are traditionally used to cure Diarrhea. It is caused by Escherichia coli. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that caffeic acid can effectively deactivate the Shikimate dehydrogenase enzyme thereby interrupting the life cycle of the organism.