Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Alkylpyrazines are important heterocyclic compounds used as flavorants in food and beverage industries. In this study, a regioselective semibiocatalytic process was developed to synthesize 2-ethyl-3,5-dimethylpyrazine (235-EDMP) over its 3-ethyl-2,5-dimethyl pyrazine (325-EDMP) isomer and vice versa. We initially explored how sterics could direct the coupling orientations between diamines and diketones to access 235- or 325-EDMP selectively. Also, the physical parameters of the reaction conditions were changed, such as reduced temperature, the order-of-addition of the reactants, and supplementation with chiral zeolites to template the orientation of the coupling partners to direct the reaction regiochemistry. Each reaction trial resulted in 50:50 mixtures of the EDMP isomers. An alternative approach was explored to control the regioselectivity of the reactions; α-hydroxy ketones replaced the diketones as the electrophilic coupling reactant used in previous trial experiments. The hydroxy ketone reactants were made biocatalytically with pyruvate decarboxylase. The coupling reaction between 2-hydroxypentan-3-one and propane-1,2-diamine resulted in the desired 235-EDMP at >70% (∼77 mg) relative to 325-EDMP in the mixture. The 3-hydroxypentan-2-one congener was biocatalyzed and reacted with propane-1,2-diamine as a proof of principle to synthesize 325-EDMP (∼60% relative abundance, ∼73 mg) over 235-EDMP. These results suggested a mechanism that was directed by the hydroxy ketone electrophilicity and the sterics at the diamine nucleophilic centers.