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Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

ABSTRACT: The enzyme prephenate dehydratase (PDT) converts prephenate to phenylpyruvate in L-phenylalanine biosynthesis. PDT is allosterically regulated by L-Phe and other amino acids. We report the first crystal structures of PDT from Staphylococcus aureus in a relaxed (R) state and PDT from Chlorobium tepidum in a tense (T) state. The two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. Both enzymes are tetramers (dimer of dimers) in crystal and solution while a PDT dimer can be regarded as a basic catalytic unit. The N-terminal PDT domain consists of two similar subdomains with a cleft in between, which hosts the highly conserved active site. In one PDT dimer two clefts are aligned to form an extended active site across the dimer interface. Similarly at the interface two ACT regulatory domains create two highly conserved pockets. Upon binding of the L-Phe inside the pockets, PDT transits from an open to a closed conformation.

SUBMITTER: Tan K

PROVIDER: S-EPMC3366492 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

Tan Kemin K Li Hui H Zhang Rongguang R Gu Minyi M Clancy Shonda T ST Joachimiak Andrzej A

Journal of structural biology 20071129 1

The enzyme prephenate dehydratase (PDT) converts prephenate to phenylpyruvate in L-phenylalanine biosynthesis. PDT is allosterically regulated by L-Phe and other amino acids. We report the first crystal structures of PDT from Staphylococcus aureus in a relaxed (R) state and PDT from Chlorobium tepidum in a tense (T) state. The two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. Both enzymes are tetramers (dimer of dimers) in crystal and so ...[more]

PMID: 18171624

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Project description:Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheAfbr) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120?mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.

Dataset Information

Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

Publications

Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

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