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Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases.

Darrell W Cockburn1, Ryan Kibler1, Haley A Brown1

  • 1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, United States.

Journal of Structural Biology
|June 29, 2021
PubMed
Summary

Ruminococcus bromii synthesizes two distinct pullulanases, Amy10 and Amy12, crucial for digesting resistant starch. Structural and biochemical analyses reveal Amy12

Keywords:
AmylosomeProtein crystallographyPullulanaseResistant starchRuminococcus bromii

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Area of Science:

  • Enzymology
  • Structural Biology
  • Microbiology

Background:

  • Pullulanases (glycoside hydrolase family 13) degrade α1,6 glucosidic linkages in starch and related glucans.
  • Ruminococcus bromii utilizes extracellular pullulanases, Amy10 and Amy12, within the amylosome complex for resistant starch digestion.
  • Understanding these enzymes is key to comprehending nutrient breakdown in the human gut.

Purpose of the Study:

  • To conduct a comparative biochemical analysis of Ruminococcus bromii pullulanases Amy10 and Amy12.
  • To determine the X-ray crystal structures of wild-type and mutant Amy12 in complex with ligands.
  • To elucidate the structural basis for substrate recognition and catalytic mechanisms.

Main Methods:

  • Comparative biochemical assays to assess enzyme activity and specificity.
  • X-ray crystallography to determine the structures of Amy12 (wild-type and D392A mutant).
  • Co-crystallization with maltoheptaose and 6³-α-D-glucosyl-maltotriose to visualize enzyme-ligand interactions.

Main Results:

  • Amy10 exhibits higher catalytic efficiency on pullulan, exclusively cleaving α1,6 linkages.
  • Amy12 shows activity on both α1,4 and α1,6 linkages, indicating functional divergence from Amy10.
  • Structures reveal an atypical mucin-binding protein (MucBP) domain in Amy12 and detailed active site interactions with substrates.

Conclusions:

  • Amy10 and Amy12 are not redundant, possessing distinct substrate specificities crucial for the amylosome complex.
  • The structural insights into Amy12 provide a basis for understanding its role in diverse starch structure recognition.
  • The findings contribute to our knowledge of microbial carbohydrate metabolism in the human gut.