Enzyme Structure/Mechanism DOI: 10.1002/ange.201108525 Insights into the Mechanistic Pathway of the Pantoea agglomerans Phenylalanine Aminomutase** Susan Strom, Udayanga Wanninayake, NishankaDilini Ratnayake, Kevin D. Walker,* and James H. Geiger* The biosynthesis of the hybrid pep- tide-polyketide antibiotic andrimid in Pantoea agglomerans requires the function of a phenylalanine amino- mutase, AdmH (designated PaPAM), which converts (S)-a-phe- nylalanine to (S)-b-phenylala- nine. [1, 2] PaPAM is a member of the class I lyase-like family that includes phenylalanine and tyrosine amino- mutases (PAMs [3] and TAMs [4, 5] , respectively), phenylalanine ammo- nia lyases (PALs), [6] tyrosine ammo- nia lyases (TALs), [7] and histidine ammonia lyases (HALs). [8] PALs, TALs, and HALs produce aryl acryl- ates from the corresponding amino- acid substrate by the elimination of ammonia. The transformations that are performed by this family of enzymes are, in part, catalyzed by a 4-meth- ylidene-1H-imidazol-5(4H)-one (MIO) prosthetic group. This cofac- tor is formed post-translationally from a tandem of active site resi- dues, typically Ala-Ser-Gly, and is believed to function as an electrophile through its a/b-unsaturated keto functional group (Scheme 1). [9] Two mechanisms for these transforma- tions have been proposed ; in the first, the amino group of the amino acid substrate acts as a nucleophile and attacks the methylidene of MIO through conjugate addition (earlier reports suggested that MIO was a dehydroalanyl moiety). [10] Ammonia is subsequently expelled from the N-alkylated substrate through an a/b-elimination process, which results in the formation of an acrylate reaction intermediate that is released as such in the ammonia lyase reaction. [11] Alterna- tively, the acrylate remains in the active site for amino group rebound to form the b-amino acid product in the amino- mutase reaction. [3, 12] A second proposed mechanism suggests that p-electrons at the ortho-carbon atom of the phenyl ring of the substrate attack MIO, which acts as a Lewis acid, by Friedel–Crafts-like activation. [6, 13, 14] The second process has been principally assigned to ammonia lyase reactions that yield unsaturated products by a/b-elimination, [6, 8, 13] but has also been implicated in the aminomutase reactions. [15] The structures of several enzymes of the MIO-dependent family were characterized in earlier reports. [16] The structure of Rhodobacter sphaeroides tyrosine ammonia lyase (RsTAL) in complex with the competitive inhibitor 2-aminoindan-2- phosphonic acid, which is covalently bound by its amino group to MIO in the active site (Protein Data Bank (PDB) 2O7E) was determined. [17] Yet, based on this structure, one of the MIO-based mechanisms was not suggested for the Scheme 1. Two proposed mechanisms for the conversion of substrate to product in a generic aminomutase: a) amino-group alkylation pathway and b) Friedel–Crafts aryl-alkylation pathway. An ammonia lyase reaction terminates at trans-cinnamate or trans-coumarate (X = H (phenylalanine) or OH (tyrosine), respectively). [*] S. Strom, [+] U. Wanninayake, [+] N. D. Ratnayake, Prof. K. D. Walker, Prof. J. H. Geiger Department of Chemistry, Michigan State University East Lansing, MI 48824 (USA) E-mail: walker@chemistry.msu.edu geiger@chemistry.msu.edu Prof. K. D. Walker Department of Biochemistry, Michigan State University (USA) [ + ] These authors contributed equally to this work. [**] We thank the Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Divi- sion (DE-FG02-06ER15822 to J.H.G.) and the National Science Foundation (CAREER Award 0746432 to K.D.W.) for financial support, and acknowledge Prof. Christopher Walsh, Harvard Medical School, for providing the cDNA clone encoding AdmH (PaPAM). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201108525. . Angewandte Zuschriften 2952  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 2952 –2956