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We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.Triphenylmethane dyes are aromatic xenobiotic compounds that are used extensively in many industrial processes, such as textile dyeing, paper printing, and food and cosmetic manufacture (11). Studies of the biodegradation of triphenylmethane dyes have focused primarily on the decolorization of dyes via reduction reactions. Several triphenylmethane dye-decolorizing microorganisms have been reported and their characteristics reviewed (2). The biochemical mechanism underlying the decolorization of triphenylmethane dyes has been elucidated in fungi (2,7,20,23) but not in bacteria. Triphenylmethane dyes are decolorized by lignin peroxidase of Phanerochaete chrysosporium (7). Laccase from the extracellular fluid of Cyathus bulleri (23) and peroxidase from Pleurotus ostreatus (20) also decolorize triphenylmethane dyes. The structural genes encoding lignin peroxidase and laccase have been cloned and characterized (8, 10). Although several triphenylmethane dye-decolorizing bacteria have been isolated (2), there are no reports of specific enzymes that decolorize these dyes. The decolorization of malachite green and crystal violet by intestinal microflora and several anaerobic bacteria proceeds through enzymatic reduction to their respective leuco derivatives (12, 16). However, the enzymes involved in this reduction have not yet been isolated or characterized in their purified forms. Their amino acid sequences and other biophysical parameters remain unknown.We recently isolated a new bacterium, Citrobacter sp. strain KCTC 18061P, that has a higher decolorization capability than any microorganism repor...

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Angiogenesis is associated with growth, invasion, and metastasis of human solid tumors. Aberrant activation of endothelial cells and induction of microvascular permeability by a vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) signaling pathway is observed in pathological angiogenesis including tumor, wound healing, arthritis, psoriasis, diabetic retinopathy, and others. Here, we show that GM3 regulated the activity of various downstream signaling pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly blocked VEGF-induced neovascularization in vivo, in models including the chick chorioallantoic membrane and Matrigel plug assay. Interestingly, GM3 suppressed VEGF-induced VEGFR-2 activation by blocking its dimerization and also blocked the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2, but not with VEGF. Primary tumor growth in mice was inhibited by subcutaneous injection of GM3. Immunohistochemical analyses showed GM3 inhibition of angiogenesis and tumor cell proliferation. GM3 also resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 inhibits VEGFR-2-mediated changes in vascular endothelial cell function and angiogenesis, and might be of value in anti-angiogenic therapy.

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most β-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein–carbohydrate interaction (PCI) or lectin–carbohydrate interaction (LCI). The HE gene was transmitted to a β-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA–specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA–ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the β-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein–glycan interaction and PCI stereochemistry potentiate the SA–ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.