Structures of open (R) and close (T) states of prephenate dehydratase (PDT)—Implication of allosteric regulation by l-phenylalanine

Tan, Kemin; Li, Hui; Zhang, Rongguang; Gu, M.; Clancy, S.; Joachimiak, Andrzej

doi:10.1016/j.jsb.2007.11.009

Cited by 36 publications

(46 citation statements)

References 56 publications

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“…Since E. coli crude extract contains endogenous PDT activity derived from a bifunctional CM-PDT enzyme (Davidson et al, 1972;Zhang et al, 1998), CM activity was analyzed in the purified fraction to ensure the absence of E. coli CM-PDT contamination. Consistent with the prior report (Tan et al, 2008), PDT activity was detectable only at high enzyme concentrations and prolonged reaction times (Supplemental Figure 5). When incubated with arogenate substrate, however, the C. tepidum ADT homolog was able to produce Phe efficiently (Supplemental Figure 5).…”

Section: Tepidum Adt Homolog Converts Arogenate Into Phesupporting

confidence: 91%

“…The weak to no PPA-AT activity detected in the Synechocystis AspAT Ib enzyme (Table 1; Graindorge et al, 2014) is consistent with the presence of BCAT-type enzymes having strong PPA-AT activity with broad substrate specificity in cyanobacteria (Stenmark et al, 1974;Jensen and Stenmark, 1975;Graindorge et al, 2014). Also, the ADT homolog of C. tepidum, previously reported as PDT (Tan et al, 2008), showed 10-fold higher catalytic efficiency for ADT than PDT activity (Table 2), whereas cyanobacteria such as Synechocystis process a bifunctional CM-PDT enzyme (Kaneko et al, 1996) and lack ADT activity (Stenmark et al, 1974;Hall et al, 1982). These phylogenetic and biochemical results together suggest that both PPA-AT and ADT, and thus the arogenate Phe pathway, were acquired by a eukaryotic ancestor of plants from a bacterial lineage that gave rise to sister phyla Chlorobi and Bacteroidetes.…”

Section: Evolutionary History Of Plant Phe Biosynthesissupporting

confidence: 77%

“…The C. tepidum homolog of plant ADTs was previously reported as PDT, although only weak PDT activity was detected (Tan et al, 2008). To test if the C. tepidum ADT homolog is capable of converting arogenate into Phe, the corresponding gene was cloned and its purified recombinant enzyme (Supplemental Figure 4) was subjected to ADT and PDT assays.…”

Section: Tepidum Adt Homolog Converts Arogenate Into Phementioning

confidence: 99%

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Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

et al. 2014

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The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.

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Section: Tepidum Adt Homolog Converts Arogenate Into Phesupporting

confidence: 91%

Section: Evolutionary History Of Plant Phe Biosynthesissupporting

confidence: 77%

Section: Tepidum Adt Homolog Converts Arogenate Into Phementioning

confidence: 99%

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Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

et al. 2014

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“…Several crystal structures of ACT domains that bind small ligands have been reported. The binding site for PheH is most similar to that of bacterial prephenate dehydratase (35); only three of the six residues identified here are conserved in that protein.…”

Section: Discussionmentioning

confidence: 84%

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Zhang

Fitzpatrick

2016

Journal of Biological Chemistry

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Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. Phenylalanine hydroxylase (PheH)2 catalyzes a key step in phenylalanine catabolism, the hydroxylation of phenylalanine to tyrosine in the liver using tetrahydropterin (BH 4 ) and oxygen. A deficiency in human PheH increases the level of phenylalanine in the blood, resulting in the inherited disease phenylketonuria (PKU) (1). Thus, the activity of PheH must be tightly controlled to maintain appropriate phenylalanine levels. PheH is activated by phenylalanine and inhibited by BH 4 (2, 3). In addition, phosphorylation of PheH at Ser-16 is reported to decrease the concentration of phenylalanine required to activate PheH (4).Mammalian PheH is a homotetramer, and each monomer contains an N-terminal regulatory domain, a central catalytic domain, and a C-terminal tetramerization domain. The other two aromatic amino acid hydroxylases, tyrosine hydroxylase (TyrH) and tryptophan hydroxylase, have similar architectures.The crystal structures of the catalytic domains of all three enzymes show very similar folds and active sites (5-7), consistent with these enzymes sharing a common catalytic mechanism (8). The structures of the regulatory domains of PheH and TyrH show that both contain ACT domains (5, 9, 10), although the two enzymes are regulated differently (2, 11). To date, there is no published structure of a full-length mammalian PheH, or indeed of any eukaryotic aromatic amino acid hydroxylase, in that the available structures are of proteins lacking the N-terminal regulatory domain, much of the C-terminal tetramerization domain, or both. The structure of a dimeric form of rat PheH containing both the catalytic and regulatory domains but lacking the C-terminal 24 residues required for tetramer formation (5) has provided the present model for the structural basis for activation by phenylalanine. In this structure the...

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“…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.…”

Section: Figmentioning

confidence: 97%

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

Shlaifer

Turnbull

2016

Extremophiles

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Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.

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Structures of open (R) and close (T) states of prephenate dehydratase (PDT)—Implication of allosteric regulation by l-phenylalanine

Cited by 36 publications

References 56 publications

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

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

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