Mechanistic, Mutational, and Structural Evaluation of a Taxus Phenylalanine Aminomutase

Abstract

The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 Å resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-α-phenylalanine to the (R)-β-isomer by exchange of the NH2/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-β-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-β-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C1-Cα and Cipso-Cβ bonds 180° in the active site prior to exchange and rebinding of the NH2/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an ∼1.5-fold increase in kcat and a decrease in KM values for sterically demanding 3'-methyl-α-phenylalanine and styryl-α-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4'-methyl-α-phenylalanine compared to those for TcPAM.

The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

“…By catalyzing the conversion of (S)-a-tyrosine to (R)-b-tyrosine, rice TAM1 stereospecificity is similar to the Taxus and C. crocatus aminomutases, which produce (R)-b-phenylalanine and (R)-b-tyrosine, respectively (Walker et al, 2004;Feng et al, 2011), and distinct from those of P. agglomerans and S. globisporus, which produce (S)-b-phenylalanine and (S)-b-tyrosine (Christenson et al, 2003;Ratnayake et al, 2011). MIO-containing aminomutase enzymes are of interest for synthetic biology studies because b-phenylalanine and b-tyrosine are required for the biosynthesis of several microbial antibiotics and anticancer drugs.…”

“…Given the sequence similarity of the two genes, it is possible that LOC_Os11g48110 also encodes an aminomutase, but perhaps with some other substrate. As MIO domain-containing aminomutases that produce b-phenylalanine have been identified in Taxus and several bacterial species (Feng et al, 2011), phenylalanine also is a possible substrate for this as yet uncharacterized rice gene. Future research will determine whether cultivated rice contains not only TAM but also PAM enzymatic activity.…”

“…One class of aminomutases has structural similarity to phenylalanine aminolyase (PAL) and tyrosine aminolyase (TAL), which catalyze the deamination of L-phenylalanine and L-tyrosine, respectively, to form (E)-cinnamic acid and (E)-4-hydroxycinnamic acid. For instance, a PAL-like phenylalanine aminomutase (PAM) in Tsuga canadensis isomerizes (S)-a-phenylalanine to the (R)-b-isomer (Feng et al, 2011), and a TAL-like tyrosine aminomutase (TAM) from C. crocatus isomerizes (S)-a-tyrosine to (R)-b-tyrosine (Wanninayake and Walker, 2013). The aminolyases and the aminomutases of this enzyme family both use a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) cofactor in the active site to catalyze the reaction, either removing an amino group or shifting it to an adjacent carbon atom.…”

A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

“…Current members, whose activity has been unambiguously confirmed experimentally, include (i) two PAMs, PaPAM from Pantoea agglomerans that yields (S)-β-phenylalanine (13)(14)(15)(16) and TcPAM from Taxus canadensis that yields (R)-β-phenylalanine (17,18), and (ii) five TAMs of SgcC4 from S. globisporus (6, 7), MdpC4 from Actinomadura madurae (19), MfTAM from Myxococcus fulvus (20), and MxTAM from Myxococcus sp. Mx-BO (20), all of which afford (S)-β-tyrosine, and CmdF from Chondromyces crocatus that yields (R)-β-tyrosine (21).…”

“…Mechanistic and structural characterizations of the MIO-containing aminomutases as a family have unveiled much unique chemistry, enzymology, and structural biology (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and exploitation of these enzymes as biocatalysts has provided access to α-and β-amino acids, which are difficult to prepare by other means (4,37). However, ʟ-phenylalanine and ʟ-tyrosine remain the only two known natural substrates (3)(4)(5).…”