Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway

Abstract

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis

“…Therefore, the tetrameric species of Mtu DAH7PS is both fully active and the inhibited species involved in allosteric regulation. Secondly, chorismate mutase is activated by formation of an enzyme complex with Mtu DAH7PS across the tetramer interface, which further stabilizes the tetrameric species of Mtu DAH7PS [21, 22]. This species is also fully active and responsive to allosteric regulation of DAH7PS activity.…”

“…Moreover, Mtu DAH7PS forms a complex and shares its allostery with M . tuberculosis chorismate mutase ( Mtu CM), which acts at the branch point that connects the shikimate pathway to Phe and Tyr production [20–22]. …”

“…Crystal structures of Mtu DAH7PS in complex with different ligands showed that there are three distinct allosteric binding sites [19, 21, 22]. Two of these are located directly at the dimer and tetramer interfaces, and bind Phe and Trp respectively.…”

“…Binding of Phe and/or Tyr to their allosteric sites at or near the dimer interface of Mtu DAH7PS has been shown to destabilize the Mtu CM interaction, leading to dissociation of the hetero-octamer, and a reduction of Mtu CM activity [21, 22]. The intricate network of interconnecting binding sites displayed by Mtu DAH7PS creates a highly sophisticated allosteric system controlling both entry into the pathway for aromatic amino acid biosynthesis and branch point partitioning.…”

X-ray Scattering Studies of Protein Structural Dynamics

“…Interestingly, addition of Phe and Tyr led to the dissociation of Mtu CM from Mtu DAHP synthase and subsequent deactivation of both enzymes. 109 This result makes particular sense when one considers that chorismate mutase is the enzyme that acts on the end product of the shikimate pathway and is located at the branch point between the biosynthesis of Trp and Tyr/Phe (Figure 2). Notably, the dissociation of the complex was observed via SEC-MALS but not SAXS, which was performed on an equimolar 20 μ M sample of Mtu DAHP synthase and Mtu CM.…”