Thermal bifunctionality of bacterial phenylalanine aminomutase and ammonia lyase enzymes.

Phenylalanine aminomutases (PAMs) are 4-methylideneimidazol-5-one (MIO)-dependent enzymes that catalyze the isomerization of (S)-a-phenylalanine to give (S)or (R)-bphenylalanine, which are precursors in the biosynthesis of various natural products. Several related tyrosine aminomutases (TAMs) have also been characterized. Furthermore, the mechanistically related MIO-dependent phenylalanine, tyrosine, and histidine ammonia lyases (PALs, TALs, and HALs) catalyze the elimination of ammonia from (S)-aphenylalanine, (S)-a-tyrosine, and (S)-a-histidine to give the corresponding trans-a,b-unsaturated carboxylic acids. Two mechanisms have been proposed for the MIO-dependent enzymes (Scheme 1A): 8] An amino-MIO adduct mechanism, which is supported by crystallographic evidence, including a structure of SgTAM from Streptomyces globisporus, and an alternate Friedel–Crafts-type reaction mechanism. A recent computational study suggests that both mechanisms could operate, depending on the presence of a specific active-site Glu residue (e.g. Glu484 in the PcPAL of Petroselinum crispum), which is suggested to electrostatically attract the substrate a-amino group that favors reaction by the Friedel–Crafts-type mechanism. MIO-dependent enzymes that do not possess a Glu residue at this position are suggested to proceed by the amino-MIO adduct mechanism. Several studies have sought to discern the structural differences that govern mutase and lyase activity. However, attempts to engineer increased lyase activity from existing mutases, based on exchanging conserved sequence motifs using site directed mutagenesis, have so far proved unsuccessful. Herein we explore the factors that differentiate MIO-dependent mutase and lyase activity, with a focus on the bacterial enzymes AdmH and EncP, which despite sharing significant sequence similarity (63% overall identity and 76% similarity) are reported to have distinct catalytic functions. Enzyme AdmH from Pantoea agglomerans is a PAM, which provides (S)-b-phenylalanine required for the biosynthesis of the antibiotic andrimid (Scheme 1B).