Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis

J Biol Chem. 2023 Feb;299(2):102789. doi: 10.1016/j.jbc.2022.102789. Epub 2022 Dec 9.

Abstract

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics simulations with small-angle X-ray scattering, mutagenesis, and heterodimer generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric interdomain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

Keywords: allosteric regulation; enzyme; molecular dynamics; mutagenesis; small-angle X-ray scattering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Isopropylmalate Synthase* / chemistry
  • 2-Isopropylmalate Synthase* / genetics
  • 2-Isopropylmalate Synthase* / metabolism
  • Allosteric Regulation
  • Biocatalysis*
  • Catalytic Domain
  • Leucine* / biosynthesis
  • Leucine* / chemistry
  • Leucine* / metabolism
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Neisseria meningitidis* / enzymology
  • Neisseria meningitidis* / metabolism
  • Pliability
  • Protein Domains*
  • Protein Multimerization
  • Scattering, Small Angle
  • X-Ray Diffraction

Substances

  • 2-Isopropylmalate Synthase
  • Leucine