LPS-Dephosphorylating Cobetia amphilecti Alkaline Phosphatase of PhoA Family Divergent from the Multiple Homologues of Cobetia spp.

A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium Cobetia amphilecti KMM 296 (CmAP), was found to effectively dephosphorylate lipopolysaccharides (LPS). Therefore, the aim of this work was to perform a comprehensive bioinformatics analysis of the structure, and to suggest the physiological role of this enzyme in marine bacteria of the genus Cobetia. A scrutiny of the CmAP-like sequences in 36 available Cobetia genomes revealed nine homologues intrinsic to the subspecies C. amphilecti, whereas PhoA of a distant relative Cobetia crustatorum JO1T carried an inactive mutation. However, phylogenetic analysis of all available Cobetia ALP sequences showed that each strain of the genus Cobetia possesses several ALP variants, mostly the genes encoding for PhoD and PhoX families. The C. amphilecti strains have a complete set of four ALP families' genes, namely: PhoA, PafA, PhoX, and two PhoD structures. The Cobetia marina species is distinguished by the presence of only three PhoX and PhoD genes. The Cobetia PhoA proteins are clustered together with the human and squid LPS-detoxifying enzymes. In addition, the predicted PhoA biosynthesis gene cluster suggests its involvement in the control of cellular redox balance, homeostasis, and cell cycle. Apparently, the variety of ALPs in Cobetia spp. indicates significant adaptability to phosphorus-replete and depleted environments and a notable organophosphate destructor in eco-niches from which they once emerged, including Zostera spp. The ALP clusterization and degree of similarity of the genus-specific biosynthetic genes encoding for ectoine and polyketide cluster T1PKS, responsible for sulfated extracellular polysaccharide synthesis, coincide with a new whole genome-based taxonomic classification of the genus Cobetia. The Cobetia strains and their ALPs are suggested to be adaptable for use in agriculture, biotechnology and biomedicine.

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with ß-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.