No abstract

A 3,5-dihydro-5-methylidine-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (TAM) isolated from the rice plant Oryza sativa (OsTAM) makes β-tyrosine (75%) and p-coumarate (25%) from α-tyrosine. OsTAM is the first TAM to have, although slight, native phenylalanine aminomutase (PAM) activity (3% relative to TAM activity). The active sites of OsTAM and a TcPAM from Taxus plants differ by only two residues (Y125 and N446 of OsTAM vs C107 and K427 of TcPAM) positioned similarly near the aryl ring of their substrates. The kinetic parameters and substrate selectivity were measured for OsTAM single mutants Y125C and N446K OsTAM and double mutant Y125C/N446K OsTAM. Compared with OsTAM, each single mutant was slower at converting α-tyrosine to its β-isomer and p-coumarate; the double mutant did not produce any detectable product. Each mutant bound α-phenylalanine ∼9-fold better than did OsTAM, suggesting that the mutations made the catalysts more selective for phenylalanine. The total turnover rate (k cat β‑Phe + k cat cinn) of each mutant for converting α-phenylalanine to both β-phenylalanine and cinnamate was ∼4-fold greater than the OsTAM 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 OsTAM.