Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein

Kyndt, John A.; Meyer, Terrance E.; Cusanovich, M.A.; Beeumen, Jozef J. Van

doi:10.1016/s0014-5793(02)02272-x

Cited by 158 publications

(138 citation statements)

References 16 publications

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“…The recombinant protein was purified to near homogeneity by His-Select nickel affinity chromatography and shown to possess PAM activity by cofactor-independent conversion of ␣-phenylalanine to ␤-phenylalanine, as determined by GC-MS analysis of the derived N-acetyl methyl ester. The calculated K m value (Michaelis-Menten plotting and non-linear regression curve fitting (R 2 ϭ 0.97) (KALEIDAGRAPH version 3.08) for the recombinant enzyme was ϳ45 Ϯ 8 M for 2S-␣-phenylalanine, with a k cat value of 0.015 s Ϫ1 and a pH optimum at approximately pH 8.5, which is similar to the pH optima reported for TAM (21) and aromatic ammonia lyases (36). PAM seems to be highly specific for 2S-␣-phenylalanine as a substrate; neither 2R-␣-phenylalanine, S-␣-tyrosine, nor S-␣-aminocyclohexanepropanoic acid hydrate was detectably isomerized to the corresponding ␤-isomer by this recombinant enzyme.…”

Section: Resultssupporting

confidence: 69%

Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

Walker

Klettke

Akiyama

et al. 2004

Journal of Biological Chemistry

127

140

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Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by phenylalanine aminomutase (PAM). A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase. A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus The final stages of Taxol biosynthesis (see Fig. 1A) in yew species involve the assembly and attachment to C-13 of the taxane core of the N-benzoyl phenylisoserinoyl side chain, which is an important pharmacophoric descriptor of this anticancer drug (1, 2). In the current practice of Taxol production, this side chain is attached by chemical semisynthesis to baccatin III, which is derived from 10-deacetylbaccatin III, a Taxus (yew) metabolite that is much more readily available than Taxol itself (3-5). In the biosynthetic pathway, five steps are involved in the construction of the side chain. The first step is considered to be the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by an aminomutase that catalyzes an intramolecular migration of the amino group and a partial internal transfer of the pro-3S hydrogen (6, 7). This step is seemingly followed by the ligase-mediated activation to the corresponding CoA ester and then the transfer of ␤-phenylalanoyl to the C-13 hydroxyl of baccatin III. The resulting intermediate (designated ␤-phenylalanoyl baccatin III or N-debenzoyl-2Ј-deoxytaxol) then likely undergoes cytochrome P450-mediated hydroxylation at the side chain 2Ј-position to generate the isoserinoyl moiety and final N-benzoylation of this side chain (8) to complete the biosynthesis of Taxol. cDNAs encoding the two transferases involved in C-13 side chain assembly have been described previously (8,9).The relative abundance of ␤-phenylalanine and side chaindeficient late pathway metabolites such as baccatin III and 10-deacetylbaccatin III in vivo (10) suggests that either the CoA ligase for ␤-phenylalanine or the CoA ester-dependent ␤-phenylalanoyltransferase (9), both of which function downstream of the aminomutase, may be rate limiting in side chain assembly and, thus, in Taxol biosynthesis. Because the phenylalanine aminomutase (PAM) 1 catalyzes the first step of the side chain assembly process and shares its primary metabolite substrate, phenylalanine, with several competing, non-taxoid phenylpropanoid pathway enzymes in plants (11-13), it is therefore an important target for genetic engineering in yew or derived cell cultures to increase Taxol production yields. PAM is also of interest enzymologically because the reaction that is catalyzed is unusual, and, in addition to the adenosylcobalamin-dependent leucine 2,3-aminomutase from Andrographis paniculata and potato tubers (14 -16), it is the only other aminomutase of plant origin d...

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Section: Resultssupporting

confidence: 69%

Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

Walker

Klettke

Akiyama

et al. 2004

Journal of Biological Chemistry

127

140

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“…The encP gene encodes a 522-amino-acid protein that is considerably smaller than eukaryotic PALs by nearly 200 amino acid residues. Although sequence homologous to plant PALs such as from Petroselinum crispum (19) (CAA57056; 30% identical and 48% similar), it rather shares greater homology to bacterial histidine ammonia lyases (HALs; EC 4.3.1.3) such as from Pseudomonas putida (21) (A35251; 36% identical and 54% similar) and to tyrosine ammonia lyase from Rhodobacter capsulatus (13) (Fig. 2).…”

mentioning

confidence: 99%

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

Xiang¹,

Moore

2005

J Bacteriol

109

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The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium "Streptomyces maritimus" is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid L-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for L-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by ϳ200 amino acid residues.

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“…A partially purified enzyme with an estimated molecular mass of 226 kDa was shown to behave similarly to PALs isolated from plants and fungi (19). More recently, a bacterial tyrosine ammonia-lyase from Rhodobacter capsulatus was characterized and shown to be 150 times more catalytically efficient toward L-tyrosine than L-phenylalanine as the substrate (20). Like EncP, there are two amino acid substitutions in the conserved active site residues in the R. capsulatus tyrosine ammonia-lyase when compared with HAL enzymes, His-83 to Leu and Glu-414 to Gln (Fig.…”

Section: Discussionmentioning

confidence: 99%

Inactivation, Complementation, and Heterologous Expression ofencP, a Novel Bacterial Phenylalanine Ammonia-Lyase Gene

Xiang

Moore

2002

Journal of Biological Chemistry

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The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) 1 is a ubiquitous higher plant enzyme that catalyzes the nonoxidative deamination of the primary amino acid L-phenylalanine to trans-cinnamic acid. Its product is the precursor of several important classes of plant phenylpropanoids including lignins, flavonoids, and coumarins. These cinnamate-derived natural products are relatively nonexistent in bacteria; however, because of the rarity of PAL in prokaryotes.Bacteria and animals do carry out a similar enzymatic reaction, the conversion of L-histidine to trans-urocanic acid by the highly homologous histidine ammonia-lyase (HAL, EC 4.3.1.3). The recent crystal structure of HAL from Pseudomonas putida (1) along with numerous biochemical studies on both enzymes (2, 3) established the reaction mechanism of this novel deamination reaction. The structure revealed that the novel prosthetic group 4-methylidene imidazol-5-one (4, 5), which is formed autocatalytically from the cyclization and dehydration of the active site tripeptide Ala-Ser-Gly, serves as the essential catalytic electrophile of this Friedel-Crafts-type enzymatic reaction (6). Although HALs and PALs have analogous mechanisms of action, PALs are considerably larger homotetrameric proteins (312 versus 215 kDa), suggesting that these related enzymes evolved independently.We recently cloned and sequenced the putative PAL gene encP, which is associated with the enterocin biosynthetic gene cluster, from the sediment-derived bacterium "Streptomyces maritimus" (7,8).2 Although the product of encP is more homologous in sequence and size to bacterial HALs than to plant PALs, circumstantial evidence suggested that EncP functions as a PAL. The bacteriostatic agent enterocin is biosynthesized by a unique type II polyketide synthase system that utilizes a novel cinnamate-derived benzoyl-coenzyme A (CoA) starter unit (Fig.

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Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein

Cited by 158 publications

References 16 publications

Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

Inactivation, Complementation, and Heterologous Expression ofencP, a Novel Bacterial Phenylalanine Ammonia-Lyase Gene

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