Dataset Information

Functional analysis of family GH36 ?-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont.

ABSTRACT: Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an ?-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14-21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for ?-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the ?-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with ?-(1,6) regioselectivity, leading to the formation of product structures up to [Hex](12) and [Hex](8), respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

SUBMITTER: Cervera-Tison M

PROVIDER: S-EPMC3485720 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Functional analysis of family GH36 α-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont.

Cervera-Tison M M Tailford L E LE Fuell C C Bruel L L Sulzenbacher G G Henrissat B B Berrin J G JG Fons M M Giardina T T Juge N N

Applied and environmental microbiology 20120824 21

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14-21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of uniq ...[more]

PMID: 22923411

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Project description:Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA∗). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA∗ was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA∗ per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA∗, this amount would generate a final yield of approximately 1-2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.

Dataset Information

Functional analysis of family GH36 ?-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont.

Publications

Functional analysis of family GH36 α-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont.

Similar Datasets

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