Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (␤-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of ␤(2,6) linkages.

Background: Invertase is a fundamental enzyme for sugar metabolism in yeast and a classical model in early biochemical studies. Results: Invertase shows an unusual octameric quaternary structure composed of two types of dimers. Conclusion: A peculiar pattern of monomer assembly through non-catalytic domain interactions determines invertase specificity. Significance: Unraveling the structural features that rule enzyme modularity casts new light on protein-carbohydrate recognition.

SummaryThe present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deepsea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to polyextremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.

Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct α/β hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.