Isoleucine binding and regulation of Escherichia coli and Staphylococcus aureus threonine dehydratase (IlvA)

Threonine deaminase (IlvA) is the first enzyme in the isoleucine branch of the branched-chain amino acid biosynthetic pathway. Kinetic studies of isoleucine feedback inhibition of Escherichia coli IlvA (EcIlvA) introduced the concept of allosteric regulation in enzymes. While the crystal structure of EcIlvA shows a tetrameric assembly of protomers with distinct catalytic and regulatory domains, the isoleucine binding site within the regulatory domain (EcIlvA_R) and its regulatory mechanism remained undefined. Here, we present the high-resolution crystal structure of the EcIlvA_R•Ile complex, identifying the isoleucine binding site. The EcIlvA_R•Ile complex reveals that isoleucine binding induces a conformational change in Phe352, propagating a 23 Å shift down the regulatory domain. We propose this conformational shift forms an allosteric pathway that extends to the adjacent protomer’s active site, mediating allosteric regulation across the protomer-protomer interface. Supporting this, the EcIlvA(F352A) mutant, though binding isoleucine with high affinity, is not inhibited due to the absence of the Phe352 sidechain. Additionally, Staphylococcus aureus IlvA (SaIlvA) is not feedback-regulated by isoleucine. Structural analysis of the SaIlvA regulatory domain reveals a different subdomain organization, preventing isoleucine binding. These findings enhance our understanding of IlvA’s allosteric regulation and offer opportunities for engineering feedback-resistant IlvA variants for biotechnological applications.