Mutation of Aryl Binding Pocket Residues Results in an Unexpected Activity Switch in an <i>Oryza sativa</i> Tyrosine Aminomutase

A 3,5-dihydro-5-methylidine-4<i>H</i>-imidazol-4-one (MIO)-dependent tyrosine aminomutase (TAM) isolated from the rice plant <i>Oryza sativa</i> (<i>Os</i>TAM) makes β-tyrosine (75%) and <i>p</i>-coumarate (25%) from α-tyrosine. <i>Os</i>TAM is the first TAM to have, although slight, native phenylalanine aminomutase (PAM) activity (3% relative to TAM activity). The active sites of <i>Os</i>TAM and a <i>Tc</i>PAM from <i>Taxus</i> plants differ by only two residues (Y125 and N446 of <i>Os</i>TAM vs C107 and K427 of <i>Tc</i>PAM) positioned similarly near the aryl ring of their substrates. The kinetic parameters and substrate selectivity were measured for <i>Os</i>TAM single mutants Y125C and N446K <i>Os</i>TAM and double mutant Y125C/N446K <i>Os</i>TAM. Compared with <i>Os</i>TAM, each single mutant was slower at converting α-tyrosine to its β-isomer and <i>p</i>-coumarate; the double mutant did not produce any detectable product. Each mutant bound α-phenylalanine ∼9-fold better than did <i>Os</i>TAM, suggesting that the mutations made the catalysts more selective for phenylalanine. The total turnover rate (<i>k</i>_cat^β‑Phe + <i>k</i>_cat^cinn) of each mutant for converting α-phenylalanine to both β-phenylalanine and cinnamate was ∼4-fold greater than the <i>Os</i>TAM rate for making β-phenylalanine and cinnamate. This switch in catalytic activity from an MIO tyrosine aminomutase (TAM) to a phenylalanine ammonia lyase (PAL) with a change of only two active site side chains suggests that these residues not only play a central role in substrate selectivity but, in part, also set the intrinsic reactivity of <i>Os</i>TAM