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Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant

Weidong Liu1, Rey-Ting Guo, Xi Chen

  • 1Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308 (China).

Chembiochem : a European Journal of Chemical Biology
|March 11, 2015
PubMed

Insights

Site-directed mutagenesis enhanced meso-diaminopimelate dehydrogenase (DAPDH) activity for D-amino acid synthesis. Structural analysis revealed the molecular basis for this expanded substrate specificity in the mutant enzyme.

Area of Science:

  • Enzymology
  • Structural Biology
  • Biochemistry

Background:

  • Meso-diaminopimelate dehydrogenase (DAPDH) typically exhibits low activity towards D-amino acids.
  • Previous studies have explored site-directed mutagenesis to alter enzyme function.

Purpose of the Study:

  • To engineer a meso-diaminopimelate dehydrogenase (CtDAPDH) with improved catalytic activity for D-amino acid synthesis.
  • To elucidate the structural basis for the expanded substrate specificity of the engineered enzyme.

Main Methods:

  • Site-directed mutagenesis was employed to modify Clostridium tetani E88 DAPDH (CtDAPDH).
  • Crystallography was used to determine the structures of the mutant enzyme in apo form and complexed with substrates.
  • Structural analysis focused on substrate binding within the catalytic cavity.

Main Results:

  • The engineered CtDAPDH mutant showed significantly enhanced catalytic ability for D-amino acid synthesis.
  • Structural data revealed meso-diaminopimelate (meso-DAP) binding outside the catalytic cavity, suggesting a two-step mechanism.
  • D-leucine (D-leu) and 4-methyl-2-oxopentanoic acid (MOPA) bound to Leu154 and Gly155 in the open enzyme form.

Conclusions:

  • Site-directed mutagenesis can effectively enhance the substrate specificity of meso-diaminopimelate dehydrogenase.
  • The structural insights provide a molecular understanding of the expanded substrate range in the mutant enzyme.
  • This work contributes to the development of enzymes for specific D-amino acid synthesis.
Keywords:
aminationdehydrogenasesenzyme catalysisprotein structuresstructural biology

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