Biosynthesis of cinnamamide and detection of phenylalanine ammonia-lyase in <i>Streptomyces verticillatus</i>

Bezanson, G. S.; Desaty, D.; Emes, Alan V.; Vining, L. C.

doi:10.1139/m70-026

Cited by 44 publications

(19 citation statements)

References 7 publications

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“…Although PAL is a ubiquitous higher-plant enzyme that catalyzes the nonoxidative deamination of phenylalanine to cinnamic acid in the committed step to phenylpropanoid metabolites (6), it has only been encountered in a few bacteria, where it is involved in benzoyl-CoA biosynthesis in "S. maritimus" (24) and Sorangium cellulosum (10) and in the biosynthesis of cinnamamide in Streptomyces verticillatus (2). We previously characterized the first prokaryotic PAL-encoding gene (encP) and showed that its inactivation resulted in the abolishment of de novo cinnamic acid and enterocin synthesis in "S. maritimus" (12,24).…”

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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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confidence: 99%

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

Xiang¹,

Moore

2005

J Bacteriol

109

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“…PAL is an essential enzyme for cinnamate and 4-coumarate production from phenylalanine. Although PAL activity in Streptomyces verticillatus was reported more than 30 years ago (3,12), no gene encoding a PAL homologue is present in the genome of S. coelicolor A3(2). S. coelicolor A3(2) has a histidine ammonia lyase (histidase), an enzyme closely related to PAL, but PAL activity of the enzyme has not been reported.…”

Section: Discussionmentioning

confidence: 99%

Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium Streptomyces coelicolor A3(2)

2003

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4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30°C, respectively. The K m value for 4-coumarate and k cat were determined as 131 ؎ 4 M and 0.202 ؎ 0.007 s ؊1 , respectively. The K m value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K m , 190 ؎ 2 M; k cat , 0.475 ؎ 0.012 s ؊1 ), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala 294 and Ala 318 are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.The enzyme 4-coumarate:coenzyme A (CoA) ligase (4CL; EC 6.2.1.12) catalyzes the last reaction of the general phenylpropanoid pathway in plants. This pathway channels carbon flow from primary metabolism to different branch pathways of secondary phenolic metabolism via the sequential action of the enzymes phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase (4CL). PAL catalyzes the conversion of phenylalanine to cinnamate, and cinnamate 4-hydroxylase catalyzes the subsequent 4-hydroxylation of cinnamate to 4-coumarate. Finally, 4CL catalyzes the conversion of 4-coumarate (4-hydroxycinnamate) and other substituted cinnamates, such as caffeate (3,4-dihydroxycinnamate) and ferulate (3-methoxy-4-hydroxycinnamate), into the corresponding CoA thiol esters, which are used for the biosynthesis of numerous phenylpropanoid-derived compounds, such as lignins, lignans, suberins, flavonoids, isoflavonoids, and various small phenolic compounds. These compounds serve diverse functions in plants, including mechanical support and rigidity in cell walls, attractants for insect pollinat...

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“…Although ubiquitous in plants and found in some fungi, PAL activity has only once before been identified in a bacterium where it appears to catalyze the first step in the biosynthesis of cinnamamide in the actinomycete Streptomyces verticillatus (18). 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).…”

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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Biosynthesis of cinnamamide and detection of phenylalanine ammonia-lyase in Streptomyces verticillatus

Cited by 44 publications

References 7 publications

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium Streptomyces coelicolor A3(2)

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

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