Mannopeptimycin, a potent drug lead, has superior activity against difficult-to-treat multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). (2S,3S)-beta-Methylphenylalanine is a residue in the cyclic hexapeptide core of mannopeptimycin, but the synthesis of this residue is far from clear. We report here on the reaction order and the stereochemical course of reaction in the formation of (2S,3S)-beta-methylphenylalanine. The reaction is executed by the enzymes MppJ and TyrB, an S-adenosyl methionine (SAM)-dependent methyltransferase and an (S)-aromatic-amino-acid aminotransferase, respectively. Phenylpyruvic acid is methylated by MppJ at its benzylic position at the expense of one equivalent of SAM. The resulting beta-methyl phenylpyruvic acid is then converted to (2S,3S)-beta-methylphenylalanine by TyrB. MppJ was further determined to be regioselective and stereoselective in its catalysis of the formation of (3S)-beta-methylphenylpyruvic acid. The binding constant (K(D)) of MppJ versus SAM is 26 microM. The kinetic constants with respect to k(cat Ppy) and K(M Ppy), and k(cat SAM) and K(M SAM) are 0.8 s(-1) and 2.5 mM, and 8.15 s(-1) and 0.014 mM, respectively. These results suggest SAM has higher binding affinity for MppJ than Ppy, and the C--C bond formation in betamPpy might be the rate-limiting step, as opposed to the C--S bond breakage in SAM.