Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme

Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution... Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase ( ef0361 ; Ef Chi18A) and a family 33 CBM ( c arbohydrate b inding m odule) ( ef0362 ; Ef CBM33A) that catalyze chitin conversion in vitro . Various types of enzyme activity assays showed that Ef Chi18A has functional properties characteristic of an endochitinase. Ef CBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of Ef CBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that Ef CBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens . Metal-depletion studies showed that Ef CBM33A is a copper enzyme. In the presence of an external electron donor, Ef CBM33A boosted the activity of Ef Chi18A, and combining the two enzymes led to rapid and complete conversion of β-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme

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Publisher
Elsevier
Copyright
Copyright © 2011 Elsevier Ltd
ISSN
0022-2836
D.O.I.
10.1016/j.jmb.2011.12.033
Publisher site
See Article on Publisher Site

Abstract

Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase ( ef0361 ; Ef Chi18A) and a family 33 CBM ( c arbohydrate b inding m odule) ( ef0362 ; Ef CBM33A) that catalyze chitin conversion in vitro . Various types of enzyme activity assays showed that Ef Chi18A has functional properties characteristic of an endochitinase. Ef CBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of Ef CBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that Ef CBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens . Metal-depletion studies showed that Ef CBM33A is a copper enzyme. In the presence of an external electron donor, Ef CBM33A boosted the activity of Ef Chi18A, and combining the two enzymes led to rapid and complete conversion of β-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field.

Journal

Journal of Molecular BiologyElsevier

Published: Feb 17, 2012

References

  • Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583
    Leisner, J.J.; Larsen, M.H.; Ingmer, H.; Petersen, B.O.; Duus, J.O.; Palcic, M.M.
  • Cleavage of cellulose by a CBM33 protein
    Forsberg, Z.; Vaaje-Kolstad, G.; Westereng, B.; Bunaes, A.C.; Stenstrom, Y.; Mackenzie, A.
  • Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8
    Moser, F.; Irwin, D.; Chen, S.L.; Wilson, D.B.
  • Coot: model-building tools for molecular graphics
    Emsley, P.; Cowtan, K.
  • A short history of SHELX
    Sheldrick, G.M.

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