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The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis alpha-isopropylmalate synthase

Frances H A Huisman1, Michael F C Hunter, Sean R A Devenish

  • 1Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand; Department of Chemistry, University of Canterbury, Christchurch, New Zealand.

Biochemical and Biophysical Research Communications
|February 2, 2010
PubMed

Insights

Alpha-isopropylmalate synthase (alpha-IPMS) in Neisseria meningitidis is crucial for leucine biosynthesis. Its regulatory domain is essential for catalytic activity, offering new avenues for drug development.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Alpha-isopropylmalate synthase (alpha-IPMS) is key in leucine biosynthesis for pathogenic bacteria like Neisseria meningitidis.
  • NmeIPMS, the enzyme from N. meningitidis, is a homodimer catalyzing the condensation of alpha-ketoisovalerate and acetyl CoA, inhibited by leucine.
  • Unlike other alpha-IPMS enzymes, NmeIPMS is activated by metal ions such as Cd(2+) and Zn(2+).

Purpose of the Study:

  • To investigate the role of the allosteric leucine-binding domain in NmeIPMS function.
  • To characterize a truncated form of NmeIPMS lacking the regulatory domain.
  • To explore the implications for inhibitor design against NmeIPMS.

Main Methods:

  • Purification and characterization of NmeIPMS.
  • Comparison of wild-type (WT) and truncated NmeIPMS.
  • Enzyme kinetics assays using alternate substrates and metal ions.
  • Assessment of catalytic activity (aldol reaction and AcCoA hydrolysis).

Main Results:

  • Loss of the regulatory domain in truncated NmeIPMS abolished the aldol reaction.
  • The truncated enzyme retained slow acetyl CoA hydrolysis activity, similar to WT.
  • Metal ion activation of NmeIPMS was confirmed, with previously inhibitory ions showing activation.

Conclusions:

  • The regulatory domain of NmeIPMS is essential for its catalytic function, not just regulation.
  • This domain may play a role in positioning catalytic residues within the TIM barrel structure.
  • Understanding the domain's importance provides potential targets for novel antimicrobial drug strategies.

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