Evolutionarily Distinct Versions of the Multidomain Enzyme α-Isopropylmalate Synthase Share Discrete Mechanisms of V-Type Allosteric Regulation

Abstract

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.

Crystal structure of an assembly intermediate of respiratory Complex II

“…One could envision that if different regulatory subunits can modulate the substrate selectivity or mechanism of an enzyme scaffold, these could eventually fuse, resulting in a more selective enzyme. A multidomain architecture with a conserved catalytic domain and a variable regulatory domain has been noted as a hallmark of several major superfamilies, including those that, like FrdA/SdhA, are arranged around a Rossmann fold 41 – 43 . A fusion with SdhE or the Fe:S subunit is not anticipated for the Complex II family because correct function requires that both the flavinylation reaction and the succinate/fumarate reaction proceed sequentially in the same molecule.…”

Principles of Allosteric Interactions in Cell Signaling

“…How to integrate the current knowledge of allostery at the molecular level across proteins and pathways in the living cell requires collaborative efforts of chemists, biophysics, and systems biologists, for example, to build allo-networks, which depict allostery at the cellular level. Lastly, we behooves us to mention that the concept of allostery has been used beyond the living cell by chemists, including the allosteric modulation of supramolecular chirality in an artificial self-assembled system, 123 biomimetic molecular allosteric analogues in chemosensors to amplify signaling, 124 to incorporate allosteric regulation in organometallic catalysts, 125 as well as in non-cooperative receptors, 126 to design allosterically tunable switches by heavy metals, 127 and to narrow the dynamic range of aptamer-based sensors. 128 …”

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis ofArabidopsismethylthioalkylmalate synthase 1

“…Several whole genome and local tandem duplications combined with subsequent deletions have resulted in a poor resolution of the MAM family in Brassicaceae. Furthermore, predicting activity from sequence data alone can be difficult and imprecise [25], as evident from IPMS enzymes, which show nearly identical substrate specificity despite having a relatively moderate sequence identity [18]. Understanding how the substrate specificity of MAMs controls the iterative chain elongation process is important from a basic science as well as a bioengineering perspective.…”