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

Abstract

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.

Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium “ Streptomyces maritimus ”

“…DNA fragments containing the targeted genes were PCR amplified (Table 2), cloned into the E. coli-streptomycete conjugal transfer vector pKC1139, and conjugated into "S. maritimus" as previously described for the construction of the related encP mutant "S. maritimus" KP (20). The singlecrossover mutants were selected after propagating transconjugants on SGGP (21) plates at 37°C, and apramycin-resistant colonies were confirmed by Southern hybridization with biotinylated gene probes.…”

“…These genes are arranged on four transcripts neighboring the enterocin polyketide synthase (PKS) genes encA, encB, and encC. We previously characterized the unique phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) gene encP and showed that its inactivation resulted in the abolishment of de novo cinnamic acid and enterocin synthesis (20). Enterocin biosynthesis could be restored in the encP-inactivated mutant "S. maritimus" KP through supplementation with cinnamic or benzoic acid as well as complementation with plasmid-borne encP.…”

Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase

“…Recently, a bacterial PAL was detected in Streptomyces maritimus, which uses the produced trans-cinnamic acid as a precursor for the antibiotic enterocin (Xiang and Moore, 2002). Again, sequence alignments revealed that the characteristic insertions were missing, indicating a closer relationship to HAL than to the plant and fungi PAL.…”

Possibility of Bacterial Recruitment of Plant Genes Associated with the Biosynthesis of Secondary Metabolites

“…The only case in which prokaryotic PAL activity has been clearly correlated to a biosynthetic protein is EncP from S. maritimus, which is needed for enterocin biosynthesis (Xiang and Moore, 2002). Although this enzyme shows higher similarities in size and sequence to prokaryotic HALs than to plant PALs (which is also the case for the above mentioned gene in S. aurantiaca), Phe was unambiguously proven as the substrate of EncP by heterologous expression of encP and production of cinammic acid in Streptomyces coelicolor.…”