Crystal Structure of Prephenate Dehydrogenase from Aquifex aeolicus

Abstract

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyperthermophile Aquifex aeolicus in complex with NAD ؉ . This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg 250 . In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser 126 and Lys 246 and the catalytic histidine, His 147 . Analysis of the structure further reveals that two secondary structure elements, ␤3 and ␤7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.The biosynthesis of tyrosine is of critical importance for the growth and survival of enteric bacteria, yeasts, fungi, and plants. Like the other aromatic amino acids, tyrosine plays a dual role in the biochemistry of the organism, acting as both a product and a precursor. In the former case, tyrosine is required for protein synthesis, whereas, in the latter, it is a substrate for enzymes in downstream metabolic pathways. The aromatic metabolites derived from tyrosine include quinones (1, 2), cyanogenic glycosides (3), alkaloids (4, 5), flavonoids (6), and phenolic compounds derived from the phenylpropanoid pathway (6, 7). Since many of these compounds are involved in primary biological processes, they are essential for viability. In plants, for example, flavonoids are important for normal development, since they are involved in auxin transport (8 -10), pollen germination (8,11,12), and signaling to symbiotic microorganisms (8, 13).The first committed step in tyrosine biosynthesis involves the conversion of prephenate to either L-arogenate or 4-hydroxyphenylpyruvate. Enzymes in the TyrA family of dehydrogenases, which are dedicated to L-tyrosine biosynthesis (14), are classified into one of three groups, depending on their substrate specificities. Prephenate dehydrogenases (PDHs) 5 accept prephenate, arogenate dehydrogenases utilize arogenate, and cyc...

Cohesion Group Approach for Evolutionary Analysis of TyrA, a Protein Family with Wide-Ranging Substrate Specificities

“…In other words, it may be the G residue at position 163 that is conserved throughout the entire homology family rather than the G residue at position 158. While the former region has been shown to be an important active-site region in both of the X-ray crystal studies done in TyrA ␣ organisms (48,71), this appears to be a region of indel disruption in TyrA ␤ . Third, the motif 232 SHLPH 236 is highly conserved in TyrA ␣ , where it has been shown to be an important active-site region in crystallography studies (48,71 (48,71), is disrupted in TyrA ␤ , where only the equivalent of the invariant 284 R is matched (although here it can be R or K).…”

“…For example, the near-invariant DxxSxK motif spanning positions 127 to 132 in Fig. 3 has been shown to be of critical importance in both of the existing X-ray crystal studies (48,71). Four of the cohesion groups (only one of them in TyrA ␣ ) exhibit variations in this motif.…”

“…TyrA proteins exemplify a case where an N-terminal Rossmann fold and a C-terminal domain comprise a "supradomain" (72), a combination that is essential for catalysis mediated by TyrA. Sun et al (71) noted that TyrA proteins belong to the "6-phosphogluconate dehydrogenase C-terminal domain-like superfamily" in the SCOP structural classification of protein domains. This superfamily has a ubiquitous N-terminal Rossmann fold joined to a C-terminal extension that is family specific.…”

“…2. Based upon comparisons of TyrA sequences from members of the TyrA ␣ subhomology grouping with the TyrA sequences of E. coli and its closest relatives (which are all TyrA ␤ members), it was previously concluded (prior to the recognition of a distinct TyrA ␤ grouping) that the TyrA sequence of E. coli and its close relatives is distinguished from the other sequences by insertion/deletion (indel) structuring (10,71). Indel structuring refers to a general case where a protein domain makes functionally important contacts with another protein domain to which it is fused.…”

“…This motif is discussed in later sections of this review, where, for convenience, it is referred to as the RxxxR motif. Finally, in TyrA ␣ sequences, the motif 290 PxMWxDI 296 consists of putative active-site residues (48,71), but this region is totally disrupted in TyrA ␤ sequences.…”