Aminomutases (AMs) catalyze the isomerization of α-amino acids to their corresponding β-amino acids. A unique class of aminomutases utilizes a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group formed via post-translational modification of three consecutive residues within the active site. These MIO-dependent aminomutases likely descend from ammonia lyases, responsible for catalyzing the removal of ammonia from α-amino acids to produce the corresponding acrylates, which are important metabolites in a variety of hosts. MIO-dependent enzymes catalyze this isomerization for aromatic amino acids (i.e. Phe, Tyr, His), the products of which are vital components to several antibiotics, antifungals, and anticancer therapeutics.In 2013, a MIO-dependent tyrosine aminomutase (TAM) from Japanese rice (Oryza sativa) was discovered while searching for defense induced metabolites. The enzyme responsible for the increased β-Tyr levels they observed, OsTAM, was cloned into E. coli and studied to elucidate its isomerization mechanism and confirm the product stereochemistry. However, during the course of this investigation, we found several interesting characteristics of OsTAM that completely changed the current dogma within MIO-dependent enzyme research including (1) OsTAM releases nearly 25% p-coumarate, the corresponding acrylate of Tyr, which is over 2-fold higher than other AMs; (2) OsTAM is the first TAM derived from plants, presenting a new opportunity to compare AMs across species; and (3) OsTAM is the first TAM to catalyze the isomerization of Phe, which paves the way for further study of the residues involved in substrate selectivity. Further study of the residues near the phenyl ring of the substrate showed two key residues, Y125 and N446, that controlled both substrate selectivity and the intrinsic activity of MIO-dependent enzymes. Arguably the most studied MIO-dependent PAM, TcPAM, lies along the biosynthetic pathway towards paclitaxel, a potent chemotherapeutic. Several studies within the lab showed that TcPAM has transamination activity when supplied an amine donor and acceptor acrylate. Recently, TcPAM was shown to catalyze the ring-opening amination of exogenously supplied cinnamate epoxides to produce phenylserine, key cores of numerous biologically active compounds. Interested in the potential of scale-up of this biocatalytic scheme, we developed a batch bioreactor method to increase cell densities of TcPAM-expressing cells and therefore increase the yield of the expressed enzyme. To increase the control and convenience of this process, we adapted autoinduction media developed by Studier in a batch bioreactor system. To the best of our knowledge, this has never been optimized for overexpression of an enzyme for later biocatalysis. With this batch bioreactor system, we turned our attentions towards scale-up of phenylserine, purification from the aqueous reaction mixture, and eventual cross-coupling to create biphenyl scaffolds aimed at recent discovery of a potent inhibitor of tumor growth with this molecular architecture. Initial studies allowed us to increase phenylserine production to the low milligram scale and develop a laboratory scale method for purification using C18 resin.
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