Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

Abstract

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (␤/␣) 8 barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å . In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å . Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, 2 E.C. 2.5.1.54) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) (Fig. 1). This reaction is the first step in the shikimate pathway, which is used to synthesize chorismate, the precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, and of other important aromatic metabolites (1). The shikimate pathway is found in plants and microorganisms, and it has more recently been shown to function in apicomplexan parasites (49). As the shikimate pathway is absent in animals, the enzymes of this pathway have been identified as possible targets for the development of antimicrobial agents (2). Regulation of the catalytic activity of DAH7PS has been shown to be an important mechanism for control of cellular levels of aromatic compounds in microorganisms and plants (3).13 C NMR studies using whole cells of Escherichia coli have demonstrated that feedback inhibition of DAH7PS is the main mechanism for controlling carbon flow into the shikimate pathway (4). Different organisms employ various strategies for this feedback inhibition. E. coli, Salmonella typhimurium, and Neurospora crassa express three DAH7PS isozymes, each sensitive to a single aromatic amino acid (5, 6). In Saccharomyces cerevisiae, there are two DAH7PS isozymes; one is inhibited by phenylalanine, and the other is inhibited by tyrosine (7). Other org...

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

“…S2A) that includes such an ACT domain dimer, similar to that of the regulatory domain of tyrosine hydroxylase (28). However, other architectures are possible, such as that of the tyrosine-binding ACT domain dimer of 3-deoxyheptulosonic acid 7-phosphate synthase (30). Also, as noted above, several possible geometries are possible for the C-terminal four-helix bundle in the activated tetramer.…”

Engineering allosteric control to an unregulated enzyme by transfer of a regulatory domain

“…This latter group includes the structurally characterized enzymes from Pyrococcus furiosus (25) and Aeropyrum pernix (26). We recently demonstrated that the DAH7PS from Thermotoga maritima, structurally composed of an ACT domain fused to the N terminus of a catalytic (β/α) 8 barrel, undergoes a remarkable conformational change associated with ligand binding (27). The associated domain reorganization controls enzyme functionality by physically gating substrate access to the active site upon binding of Tyr (Fig.…”

Interdomain Conformational Changes Provide Allosteric Regulation en Route to Chorismate

“…The pathway branches at chorismate, and chorismate mutase catalyzes the formation of prephenate from chorismate, which in turn will form Tyr and Phe. mechanism for elaborating feedback inhibition on DAH7PS activity (4,7,17). The combination of DAH7PS and CM domains may represent another case of this phenomenon.…”

“…Whereas the type I␤ DAH7PS from Thermotoga maritima undergoes significant ACT domain movement associated with the binding of inhibitor Tyr (7), for other enzymes, such as the type II M. tuberculosis DAH7PS (13) and the type I␣ N. meningitidis DAH7PS (14), allosteric inhibition is conferred by more subtle structural changes and alteration in protein dynamics. The variation in allosteric machinery and mechanism for this enzyme is intriguing both from a mechanistic and evolutionary point of view.…”

“…Type I␤ enzymes are more varied and either comprise just the core catalytic barrel and show no allosteric regulation or have a discrete domain found at either the N or C termini of the barrel (6). This addition can be either an ACT domain (7,8) (named after the enzymes in which it was originally identified, aspartokinase, chorismate mutase, and TyrA) or a chorismate mutase (CM) enzyme (9), which catalyzes a branch point step of the pathway (Fig. 1).…”