Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

ABSTRACTModular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

Computational investigations of allostery in aromatic amino acid biosynthetic enzymes

Allostery, in which binding of ligands to remote sites causes a functional change in the active sites, is a fascinating phenomenon observed in enzymes. Allostery can occur either with or without significant conformational changes in the enzymes, and the molecular basis of its mechanism can be difficult to decipher using only experimental techniques. Computational tools for analyzing enzyme sequences, structures, and dynamics can provide insights into the allosteric mechanism at the atomic level. Combining computational and experimental methods offers a powerful strategy for the study of enzyme allostery. The aromatic amino acid biosynthesis pathway is essential in microorganisms and plants. Multiple enzymes involved in this pathway are sensitive to feedback regulation by pathway end products and are known to use allostery to control their activities. To date, four enzymes in the aromatic amino acid biosynthesis pathway have been computationally investigated for their allosteric mechanisms, including 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, anthranilate synthase, chorismate mutase, and tryptophan synthase. Here we review the computational studies and findings on the allosteric mechanisms of these four enzymes. Results from these studies demonstrate the capability of computational tools and encourage future computational investigations of allostery in other enzymes of this pathway.

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.