Discovery of additional members of the tyrosine aminomutase enzyme family and the mutational analysis of CmdF.

The tyrosine aminomutase (TAM) CmdF converts L-Tyr preferentially to (R)-beta-Tyr--a biosynthetic building block subsequently incorporated into the highly cytotoxic chondramides by the myxobacterium Chondromyces crocatus. Together with the similar enzymes SgcC4 from Streptomyces globisporus and MdpC4 from Actinomadura madurae, which preferentially produce (S)-beta-Tyr, CmdF belongs to a novel 2,3-aminomutase enzyme family closely related to the aromatic amino acid ammonia lyase. Although considerable insight into the underlying catalytic mechanism has been provided recently by structural and mechanistic studies, the key determinants of product specificity and stereochemical preference of TAM enzymes remain to be elucidated in detail. We report herein the discovery and heterologous expression of additional TAMs from prokaryotic sources. These studies reveal a high degree of evolutionary diversification within this expanding enzyme family. Attempts to genetically engineer CmdF to exhibit ammonia lyase-type activity by the exchange of conserved sequence motifs were largely unsuccessful. However, the variation of a semiconserved glutamic acid residue was found to impact stereoselectivity. Replacement of this residue by lysine significantly increased the enantiomeric excess of (R)-beta-Tyr from 69 to 97 % ee, while substitution with methionine promoted racemization. These results suggest that it should be possible to elucidate a mechanism for control of stereoselectivity in the TAM family by the application of directed evolution to CmdF. Furthermore, our findings indicate the potential to fine-tune the catalytic properties of TAMs for their use as biocatalysts or in engineered biosynthetic pathways.