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Microbiome: Structures from Lactic Acid Bacteria

SBKB [doi:10.1038/sbkb.2012.159]
Featured Article - September 2013
Short description: Structures of GH1-family 6-P-β-glucosidases in complex with ligands reveal determinants for substrate recognition.

Active site of 6-P-β-glucosidase (stereoview). LbPbg1 in complex with a phosphate ion and a
β-glucose moiety. Hydrogen bonds are shown as dashed lines. 1

Lactic acid bacteria are Gram-positive organisms that produce lactic acid as the major end product of carbohydrate metabolism. They are part of the human microbiome, playing a role in health and disease, and also have important industrial applications. In these bacteria, the uptake of carbohydrates across the membrane is coupled with phosphorylation of the incoming sugar. After translocation, the β-glycosidic bond in 6′-P-β-glucoside is cleaved by 6-P-β-glucosidases, releasing 6-P-β-glucose (BG6 or glycon) and the remaining portion of the substrate (aglycon). These enzymes belong to the GH1 family of hydrolysases, which show diverse enzymatic activities.

Joachimiak and colleagues (PSI MCSG) now report five crystal structures of 6-P-β-glucosidases from lactic acid bacteria, namely Lactobacillus plantarum WCFS1 (LbPbg1) in the apo form (PDB 4GZE) and in complex with phosphate and a β-glucose molecule corresponding to the aglycon molecule (PDB 3QOM), and Streptococcus mutans UA159 (SmBgl) in complex with a sulfate ion (PDB 3PN8) and with the reaction product BG6 (PDB 4F66). The structure of a mutant SmBgl enzyme in complex with 6′-P-salicin substrate was also determined (PDB 4F79).

The authors show that the L. plantarum and S. mutans enzymes can catalyze diverse glucoside substrates, but have surprisingly different kinetic properties and affinities for them. This result is in line with previous reports indicating that various lactic acid bacteria show quite different 6-P-β-glucosidase and 6-P-β-galactosidase activities. It is also possible that the preferred substrates for these enzymes have not yet been identified.

Structures were solved by single anomalous dispersion using SeMet-labeled protein crystals. 6-P-β-glucosidase is a single-domain protein, with the TIM-barrel structure typical of GH1-family members. Both proteins form homodimers in the crystals, a behavior that was confirmed in solution by size exclusion chromatography. Comparison of the new structures with those of other GH1-family enzymes showed a high level of structural homology, including conservation of the active site. Interestingly, sequence alignment of all annotated L. plantarum 6-P-β-glucosidases suggest that they share an identical glycon binding site, while the aglycon binding site and entry to their active sites vary in sequence. Moreover, the same structural elements show variability between S. mutans 6-P-β-glucosidases.

Together, these results suggest that differences in the aglycon binding site and the entrance to the active site of various 6-P-β-glucosidases may be important for determining substrate specificity.

Arianne Heinrichs

References

  1. K. Michalska et al. GH1-family 6-P-β-glucosidases from human microbiome lactic acid bacteria.
    Acta Crystallogr D Biol Crystallogr. 69, 451-463 (2013). doi:10.1107/S0907444912049608

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