J Bacteriol 187(12): 4286-4289

Biochemical Characterization of a Prokaryotic Phenylalanine Ammonia Lyase

College of Pharmacy,^{Department of Chemistry, University of Arizona, Tucson, Arizona 85721}²

^{Corresponding author. Mailing address: College of Pharmacy, University of Arizona, Tucson, AZ 85721-0207. Phone: (520) 626-6931. Fax: (520) 626-2466. E-mail:}ude.anozira.ycamrahp@eroom.

Received 2005 Jan 6; Accepted 2005 Mar 3.

Abstract

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 ∼200 amino acid residues.

Abstract

The bacteriostatic agent enterocin is a natural product of the marine bacterium “Streptomyces maritimus” whose biosynthesis involves a number of unusual features (9, 16, 17, 22). Among these is the formation of the rare polyketide synthase starter unit benzoyl-coenzyme A (benzoyl-CoA) (15). The initial biochemical reaction involves the conversion of the amino acid l-phenylalanine to trans-cinnamic acid by the novel bacterial phenylalanine ammonia lyase (PAL; EC 4.3.1.5) EncP (24). Activation of cinnamic acid to its CoA thioester, followed by a single round of β-oxidation, yields benzoyl-CoA (7, 8, 23), which primes the enterocin type II polyketide synthase for chain extension with seven molecules of malonyl-CoA (Fig. (Fig.11).

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FIG. 1.

EncP-catalyzed conversion of phenylalanine to trans-cinnamic acid and biosynthesis of benzoyl-CoA-derived enterocin in “S. maritimus.”

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). Enterocin biosynthesis could be restored in encP-inactivated mutants through supplementation with cinnamic or benzoic acid, as well as complementation with plasmid-borne encP. Furthermore, the heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor led to the production of cinnamic acid in the fermented cultures (24). Here we report the biochemical characterization of this novel bacterial PAL, including substrate specificity, pH dependence, and kinetics, as well as its inhibition with the known plant PAL inhibitor 2-aminoindan-2-phosphonic acid (AIP) (1).

Acknowledgments

This work was supported by the NIH (AI47818).

We thank Joseph P. Noel (Salk Institute for Biological Studies, La Jolla, CA) for the vector pHIS8, Jerzy Zon (Wroclaw University, Wroclaw, Poland) for generously providing the inhibitor AIP, and Yoshimitsu Hamano (University of Arizona) for helpful discussions.

Acknowledgments

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