A Monofunctional and Thermostable Prephenate Dehydratase from the Archaeon Methanocaldococcus jannaschii

Abstract

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.

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

“…It is noteworthy that Trp concatenates from upper Gammaproteobacteria and from Betaproteobacteria defined a single supercohesion group. The above-mentioned phylogenetic tree of PheA sequences, having less resolving power than Trp concatenates, nevertheless exhibited neighboring clusters, albeit with weak bootstrap support (44). A phylogenetic tree of TyrA sequences ( Fig.…”

“…6, panel 4). It seems likely that many of the latter cohesion groups will merge into a single group in view of the precedent of TrpSCG-2, the finding that PheA sequences from upper Gammaproteobacteria and Betaproteobacteria appear to join together on a phylogenetic tree (44), and the finding that common aromatic pathway proteins of upper Gammaproteobacteria and Betaproteobacteria also form single, cohesive groupings (data not shown). Indeed, seven upper Gammaproteobacteria TyrA cohesion groups and orphans (Microbulbifer degradans, TyrCG-6, TyrCG-3, TyrCG-5, TyrCG-2, Methylococcus capsulatus, and Nitrosococcus oceani) join one another at a common node to the exclusion of TyrA proteins from any other phylogenetic grouping, as is also the case for eight Betaproteobacteria cohesion groups (TyrCG-9, TyrCG-8, TyrCG-7, Thiobacillus denitrificans, Methylobacillus flagellatus, Dechloromonas aromatica, Azoarcus sp., and Chromobacterium violaceum).…”

“…Other character states of enzymes performing early aromatic pathway reactions have been noted to exhibit qualitative differences that define the same taxonomic split. Kleeb et al (44) observed two clusters on a phylogenetic tree of PheA sequences from Gammaproteobacteria (called by them Gammaproteobacteria I and Gammaproteobacteria II). The latter clusters correspond to lower Gammaproteobacteria and upper Gammaproteobacteria.…”

Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea

“…As a form of allosteric control, E. coli CM-PDT is reported to undergo an L-Phe-induced formation of higher order species (Zhang et al 1998). However, no significant shift in the molecular weight of NeTyrA was observed in the presence of 1 mM L-Phe, possibly a function of the heterogeneity of the sample, although PDT from Methanocaldococcus jannaschi also did not change its assembly in the presence of L-Phe (Kleeb et al 2006). …”

“…Helical content calculated from the primary sequences of these archaeal TyrAs using secondary structure prediction programs was between 50 and 60 %. These values are typical for many CM, PD, and PDT proteins from other organisms, including hyperthermophiles (Chiu et al 2010;Kleeb et al 2006;Lee et al 1995;Sun et al 2006;Tan et al 2008). The unfolding of IhTyrA and NeTyrA by thermal denaturation was followed by measuring changes in ellipticity at 222 nm (Fig.…”