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Related Experiment Videos

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

M Nojiri1, T Saito

  • 1Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.

Journal of Bacteriology
|November 26, 1997
PubMed
Summary
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Investigating Poly(3-hydroxybutyrate) (PHB) depolymerase structure revealed the catalytic domain is essential for PHB hydrolysis. The fibronectin type III-like and substrate-binding domains are crucial for PHB binding and degradation.

Area of Science:

  • Biochemistry
  • Enzymology
  • Polymer Science

Background:

  • Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer with potential applications.
  • PHB depolymerase from Alcaligenes faecalis T1 is a key enzyme for PHB degradation.
  • The enzyme comprises catalytic (C), fibronectin type III-like (F), and substrate-binding (S) domains.

Purpose of the Study:

  • To elucidate the functional roles of the individual domains of PHB depolymerase.
  • To investigate the contribution of specific amino acid residues to the enzyme's catalytic activity.
  • To understand the structure-function relationship of PHB depolymerase.

Main Methods:

  • Construction and analysis of domain deletion, inversion, chimera, and extra-F-domain mutants.
  • Enzyme activity assays for PHB and D-(-)-3-hydroxybutyrate trimer hydrolysis.

Related Experiment Videos

  • PHB-binding ability assessment.
  • Site-directed mutagenesis of key catalytic residues (214Asp, 273His, 139Ser).

    • Main Results:

    • Mutants lacking both F and S domains lost PHB-hydrolyzing activity but retained trimer-hydrolyzing activity and reduced PHB binding.
    • Mutants with F domain deleted or inverted S domain lost PHB hydrolysis but retained binding and trimer hydrolysis.
    • Mutants with F domain substitution or addition retained both activities and binding.
    • Mutations at 214Asp and 273His abolished all enzymatic activity.

    Conclusions:

    • The catalytic domain is essential for PHB hydrolysis.
    • The F and S domains are critical for PHB binding and hydrolysis, with the F domain playing a significant role.
    • A catalytic triad involving 139Ser, 214Asp, and 273His is essential for the enzyme's function.