Biocatalytic process-development continues to advance toward discovering alternative transformation reactions to synthesize fine chemicals. Here, a 5-methylidene-3,5dihydro-4H-imidazol-4-one (MIO)-dependent phenylalanine aminomutase from Taxus canadensis (TcPAM) was repurposed to irreversibly biocatalyze an intermolecular amine transfer reaction that converted ring-substituted transcinnamate epoxide racemates to their corresponding arylserines. From among 12 substrates, the aminomutase ringopened 3′-Cl-cinnamate epoxide to 3′-Cl-phenylserine 140 times faster than it opened the 4′-Cl-isomer, which was turned over slowest among all epoxides tested. GC/MS analysis of chiral auxiliary derivatives of the biocatalyzed phenylserine analogues showed that the TcPAM-transamination reaction opened the epoxides enantio-and diastereoselectively. Each product mixture contained (2S)+(2R)-anti (erythro) and ( 2S)+(2R)-syn (threo) pairs with the anti-isomers predominating (∼90:10 dr). Integrating the vicinal proton signals in the 1 H NMR spectrum of the enzyme-catalyzed phenylserines and calculating the chemical shift difference (Δδ) between the anti and syn proton signals confirmed the diastereomeric ratios and relative stereochemistries. Application of a (2S)-threonine aldolase from E. coli further established the absolute stereochemistry of the chiral derivatives of the diastereomeric enzymatically derived products. The 2R:2S ratio for the biocatalyzed anti-isomers was highest (88:12) for 3′-NO 2 -phenylserine and lowest (66:34) for 4′-Fphenylserine. This showed that the stereospecificity of TcPAM is in part directed by the substituent-type on the cinnamate epoxide analogue. The catalyst also converted each cinnamate epoxide analogue to its corresponding isoserine, highlighting a biocatalytic route to arylisoserines, which play a key role in building the pharmacophore seen in anticancer and protease inhibitor drugs.

A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-β-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 μM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-β- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(β) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants.

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.

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In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.