Biocatalysis of a Paclitaxel Analogue - ACS Publications - American

Oct 25, 2017 - (TBT); c) 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT); d) baccatin III 3-amino-13-O-phenylpropanoyltransferase (BAPT); e) cur...
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Article Cite This: Biochemistry XXXX, XXX, XXX-XXX

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Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N‑Debenzoyl‑N‑(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase Chelsea K. Thornburg,‡ Tyler Walter,† and Kevin D. Walker*,†,‡ †

Department of Chemistry and ‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States S Supporting Information *

ABSTRACT: 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 (MBPBAPT). 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 onepot 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 Ndebenzoylpaclitaxel 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.

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ince its approval for refractory ovarian cancer in 1992, the demand and production milestones for paclitaxel (Taxol) (1) and its derivatives have increased. Continued inclusion of paclitaxel in regimens against more cancer types, including several combination therapies and applications in heart restenosis, largely contribute to the increased demand.1−3 The primary production resources of paclitaxel to keep up with demand are Taxus plant cell fermentation (PCF)4 and semisynthetic methods.5,6 Despite these production efforts, paclitaxel and its analogues often go through cycles of shortages as shown occasionally on various drug shortages lists.7−9 These shortages linked with high demand, manufacturing challenges, shortages of raw materials, stockpiling by large hospitals, and other economic disincentives can negatively affect patient health care.9−14 Engineering portions of the pathway to 1 in bacteria or yeast have benefits over current plant-cell fermentation methods.15 Within Taxus cells, many paclitaxel biosynthetic enzymes accept multiple substrates and produce multiple products creating a complex metabolic network of over 400 taxoids stemming from the common precursor taxa-4(5),11(12)diene.16,17 Competing pathways siphon carbon flux away © XXXX American Chemical Society

Figure 1. Paclitaxel.

from (and return it to) the paclitaxel pathway within the complex metabolite pool.16,18−20 Thus, defining a linear biosynthetic route to paclitaxel is daunting.21 Concerns about flux-control, enzyme specificity, and incomplete characterization of the paclitaxel pathway18,22−25 limit efforts to Received: September 13, 2017 Revised: October 13, 2017

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DOI: 10.1021/acs.biochem.7b00912 Biochemistry XXXX, XXX, XXX−XXX

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Figure 2. Biocatalytic routes using paclitaxel pathway acyltransferases from Taxus plants and enzymes co-opted from bacteria are shown. Pathways from a putative 2-O-debenzoyl-10-O-deacetylbaccatin III (4) via precursors N-de-(t-butoxycarbonyl)docetaxel (N-DBocD) (11) and Ndebenzoylpaclitaxel (N-DBzP) (12) to docetaxel (13) and a paclitaxel analog N-debenzoyl-N-(2-furoyl)paclitaxel (N-2-FP) (14), respectively. Catalysts on the pathways include a) truncated TycA tyrocidine synthase (PheAT), ATP, CoA, and Mg2+; b) taxane-2α-O-benzoyltransferase (TBT); c) 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT); d) baccatin III 3-amino-13-O-phenylpropanoyltransferase (BAPT); e) currently unidentified N-debenzoyl-2'-deoxypaclitaxel 2′-hydroxylase (T2′OH); f) triethylamine and t-butoxycarbonyl anhydride; g) N-debenzoylpaclitaxel Nbenzoyltransferase (NDTNBT); and h) a benzoate CoA ligase (BadA), ATP, CoA, and Mg2+. Compounds: (3R)-β-phenylalanine (2); (3R)-βphenylalanyl CoA (3); (2R,3S)-phenylisoserine (PheIso) (9); (2R,3S)-phenylisoserinyl CoA (PheIso CoA) (10); 10-deacetylbaccatin III (10-DAB) (5); baccatin III (6); N-de-(t-butoxycarbonyl)-2'-deoxydocetaxel (7); N-debenzoyl-2'-deoxypaclitaxel (N-DBz2DP) (8); 2-furoic acid (15); furoyl CoA (16). Compounds (in color) and enzymatic steps (bold underlined letters) used herein are highlighted.

include taxane 5-O-acetyltransferase (TAT),28 taxane-2α-Obenzoyltransferase (TBT),31,32 10-deacetylbaccatin III 10-Oacetyltransferase (DBAT),33 baccatin III: 3-amino-13-O-phenylpropanoyl CoA transferase (BAPT),34 and N-debenzoyltaxane-N-benzoyltransferase (NDTNBT).35,36 Several accounts of paclitaxel biosynthesis propose a pathway in which BAPT transfers a (3R)-β-phenylalanyl moiety from its CoA precursor (3) to the C13 hydroxyl of baccatin III (6) to form N-debenzoyl-2′-deoxypaclitaxel (N-DBz2DP) (8). A cytochrome P450 (T2′OH) is proposed to hydroxylate the latter at the 2′-position to form a phenylisoserinyl intermediate (12) (Figure 2).27 A separate study supported the 2′hydroxylation sequence when a β-phenylalanyl CoA ligase (βPhCL) was discovered in Taxus baccata cell cultures.37 Unfortunately, this former study did not characterize β-PhCL with (2R,3S)-phenylisoserine (PheIso) (9), the side chain that gives drug efficacy to paclitaxel (1) and its many analogues.35,38

genetically engineer an organism to biocatalyze the complete linear route.26 For instance, genes encoding the enzymes for several steps remain unidentified, including a proposed C1 hydroxylase (T1βOH), C9 hydroxylase (T9αOH), C4−C20epoxidase, an oxomutase that makes the oxetane ring, a C9 oxidase, and a C2′-hydroxylase (C2′αOH).27 As another example, the regiospecificity of a Taxus taxadiene-5α-ol-Oacetyltransferase (TAT)28 switches depending on the hydroxylation pattern on the substrate. TAT acetylates the lone 5hydroxyl group of taxadiene-5α-ol, but preferentially acetylates the C9 and C10 hydroxyls of the 5,9,10,13-tetraol over the 5hydroxyl.29 Of the identified enzyme catalysts on the pathway to 1, five are functionally characterized acyltransferases from the BAHD family (named as an acronym for the first four enzymes characterized in this family)30 and proposed to play a role in paclitaxel biosynthesis in Taxus plants. These acyltransferases (Figure 2) share 57−65% sequence identity among them and B

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Biochemistry The biosynthetic pathway to 1 then finishes with N-aroylation of isoserine to produce the bioactive compound (Figure 2).36,39 While the discovery of a Taxus β-PhCL was long-awaited,37 the 2′-hydroxylase that oxidizes N-DBz2DP (8) remains elusive (Figure 2).27 Initial studies with BAPT demonstrated catalytic activity with (2R,3S)-phenylisoserinyl CoA (PheIso CoA) at a lower rate than with (3R)-β-phenylalanyl CoA (β-Phe CoA) (2).39 However, since paclitaxel analogues are biologically inactive without the 2′-hydroxyl group,40 our efforts here focused on further characterizing BAPT with PheIso CoA. We foresee building a foundation to develop a condensed, streamlined biocatalytic route to paclitaxel analogues from baccatin III (6), an abundant intermediate isolated from plant cell cultures.6,41 More important, this pathway would bypass the need to identify the C2′-hydroxylase (T2′OH) gene27 (Figure 2). In this proof-of-concept study, we used a four-step cascade reaction that contained the enzymes, PheAT (a truncated TycA module of a nonribosomal peptide synthase (NRPS), tyrocidine synthase, from Bacillus brevis),42 a broad-specificity benzoate:CoA ligase (BadA),43 a Taxus plant N-benzoyltransferase (NDTNBT), and a modified maltose binding protein fusion of BAPT (MBP-BAPT).35,39 This group of enzymes catalyzed the formation of the important intermediate Ndebenzoylpaclitaxel (N-DBzP) (12), which is a precursor of several different natural and semisynthetic bioactive taxanes,16 including N-debenzoyl-N-(2-furoyl)paclitaxel (N-2-FP) (14). The latter belongs to a family of analogues with greater efficacy than that of paclitaxel against certain carcinoma types.44,45 One example shows that an N-2-FP analogue synthetically derived from 1-deoxybaccatin-VI has 15-fold higher cytotoxicity (IC50) in hepatic carcinoma cell line H460.46

from Natland International Corporation (Research Triangle Park, NC). Additional reagents were sourced as follows: TEA (100%) (J. T. Baker, Center Valley, PA), acetic anhydride (>99.4%) and trifluoroacetic acid (>99.5%) (EMD Chemicals, Billerica, MA), di-t-butyl-dicarbonate (>99%) and ethyl chloroformate (97%) (Sigma-Aldrich, St. Louis, MO), C18 silica gel resin (carbon 23%, 40−63 μm) (Silicycle, Quebec City, Quebec, Canada). BAPT Expression and Purification. BL21(DE3) E. coli (Invitrogen, Carlsbad, CA) were engineered to express plasmid pMBP-CterBAPT that encoded a fusion protein, linking BAPT with a maltose binding protein affinity tag and a C-terminal His6-epitope (MBP-BAPT) (Figures S1−S2). A single colony of BL21(DE3) E. coli (Invitrogen, Carlsbad, CA) engineered to express pMBP-CterBAPT was selected and used to inoculate a 100 mL culture of LB media containing 100 μg/mL ampicillin. The culture was grown overnight at 37 °C. Aliquots (7 mL) of this starter culture were used to inoculate a flask of fresh LB media (1 L) containing 100 μg/mL ampicillin. Gene expression was induced with 0.1 mM IPTG when the cell density reached an OD600 of 0.6, and the cells were grown for ∼16 h at 18 °C. Bacteria were harvested by centrifugation at 8000g for 5 min. Pellets were resuspended in Buffer A (30 mM MOPS (pH 8), 300 mM NaCl, 15 mM imidazole, 5% glycerol) at 3 mL/g pellet (w/w). Phenylmethylsulfonyl fluoride (Millipore Sigma, Billerica, MA) (100 mM stock in isopropanol) was added to the cell suspension at a final concentration of 1 mM. The cell suspensions were kept on ice, lysed by sonication (Misonix XL 2020) in batches (15 g pellet/ ∼4-L culture; 8 L total) for 15 min in three cycles of 10-s pulses, followed by 20 s on ice. The lysed cells were centrifuged at 18000g for 20 min, and the supernatant was further centrifuged at ∼100000g for 1.5 h. The clarified supernatant was loaded onto a Fast-Flow Ni2+−NTA column (10 mL) (Qiagen) pre-equilibrated with Buffer A at 4 °C. The column was washed with 5 column volumes (CV) of Buffer A, and eluted with 3 CV of Buffer A containing 250 mM imidazole. The eluent was concentrated in a Millipore Amicon Ultra 30 kDa cutoff concentrator and buffer exchanged with 30 mM MOPS, pH 8.0 with 5% glycerol (Buffer B) without imidazole. The eluent was loaded onto an amylose-binding resin (1 mL), pre-equilibrated with Buffer B. The amylose column was then washed with 5 CV of Buffer B and eluted with 3 CV of Buffer B containing maltose (10 mM). The eluted MBP-BAPT was buffer-exchanged with Buffer B in a 30 kDa cutoff concentrator (Millipore Amicon) and concentrated to 1−3 mL. Aliquots (40 μL) of MBP-BAPT (∼300 μg/mL, estimated by the Coomassie protein assays, Thermo Scientific Pierce, Grand Island, NY) were flash-frozen in liquid nitrogen and stored at −80 °C. Enzyme purity was estimated by SDS-PAGE, band densitometry, and LC-MS (Figures S3−S4). MBP-BAPT Assays to Track Activity During Purification. The PheAT CoA ligase was expressed and purified (see Supporting Information and Figure S5) as previously described and used diagnostically in coupled assays to follow MBP-BAPT activity in the eluents from each column during purification. Assays were prepared on ice and incubated at 31 °C for 5 min prior to enzyme addition. The PheAT assay (200 μL) contained baccatin III (1 mM in acetonitrile, 10% (v/v)), followed by 200 mM MOPS (pH 8), 3.5 mM MgCl2, 1 mM ATP, 1 mM β-phenylalanine, 1 mM CoA, and PheAT (0.75 mg/mL). After 1 h at 31 °C, an aliquot (50 μL) from the MBPBAPT purification fraction was added and allowed to react for

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MATERIALS AND EXPERIMENTAL DETAILS Materials. Chloramphenicol (99%) and HEPES (>99%) were obtained from Fluka/Sigma-Aldrich (St. Louis, MO). MOPS (>99%) was obtained from Research Products International, Corp. (Mt Prospect, IL). ATP, DTT, isopropyl β-D-1thiogalactopyranoside (IPTG), ampicillin, kanamycin, phenylmethylsulfonyl fluoride (PMSF), and tris(2-carboxylethyl)phosphine·HCl (TCEP), cobalt-affinity chromatography resin, and were purchased from Gold Bio (St. Louis, MO). The following reagents were purchased from New England Biolabs (Ipswich, MA): dNTPs, Phusion HF DNA polymerase, all restriction enzymes, and T4 DNA ligase. The QIAGEN plasmid mini prep kit and gel extraction kit were obtained from QIAGEN (Valencia, CA). The following reagents were purchased from Promega (Madison, WI): PureYield plasmid mini purification system and Wizard SV gel and PCR clean-up system. Coenzyme A (95%) was obtained from Lee Biosolutions (St. Louis, MO). (3R)-β-Phenylalanine (98%) was purchased from Peptech (Burlington, MA). EDTA-free protease inhibitor cocktail tablets were purchased from Roche Life Sciences (Indianapolis, IN). The PfuTurbo DNA Polymerase and Escherichia coli strain BL21(DE3) were sourced from Invitrogen (Carlsbad, CA), The pET28a expression vector came from (Novagen, EMD Millipore, Billerica, MA). Coenzyme A (95%) was obtained from Lee Biosolutions (St. Louis, MO). (3R)-β-Phenylalanine (98%) was purchased from Peptech (Burlington, MA). (2R,3S)-phenylisoserine (98%) was purchased from Waterstonetech, LLC (Carmel, IN). All taxanes (baccatin III (>98%), 10-deacetyl baccatin III (>98%), docetaxel (>98%) and paclitaxel (>98%) were purchased C

DOI: 10.1021/acs.biochem.7b00912 Biochemistry XXXX, XXX, XXX−XXX

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30.00 (s, 9″), 37.57 (s, 5″, 6″), 40.43 (s, 8″), 40.66 (d, J = 7.67 Hz, 2″), 59.17 (s, 3c), 67.30 (br s, 5′), 74.09 (s, 1″), 76.32 (m, 2′, 3″, 3′), 79.24 (s, 2c), 89.60 (br s, 1′, 4′), 120.78 (s, 5), 129.87−134.79 (C4 aromatic carbons), 144.64 (s, 8), 146.91 (s, 4), 150.74 (s, 2), 152.15 (s, 6), 176.33 (s, 7″), 176.98 (s, 4″), 205.43 (s, 1c). (See Figure S9 for carbon numbering). LC-ESIMS m/z 929.0940 [M − H]−; calculated for C30H44N8O18P3S: 929.1713. MBP-BAPT Kinetic Assays. PheIso CoA (10) and β-Phe CoA (3) (see Supporting Information for synthesis34 and Figures S11−S15) (each at 10 mM stock) were dissolved separately in deionized water (pH 3 with 8.8% formic acid). Baccatin III (6) (10 mM stock) and 10-deacetylbaccatin III (10-DAB) (5) (10 mM stock) were dissolved in acetonitrile or methanol, respectively. To establish steady-state kinetic rates of MBP-BAPT with respect to enzyme concentration and time, the acyl CoA and taxane were combined in 200 mM MOPS (pH 8) containing 5% glycerol and preincubated at 31 °C for 10 min before the addition of ∼0.2 μg (0.024 nmol, 0.12 μM) MBP-BAPT (200 μL total volume). Assays were acid-quenched with 8.8% formic acid (aq) to pH 5 at different time points to generate a time-course series. Docetaxel (1 μM) was added as an internal standard. Concentrations of acetonitrile or methanol (depending on the taxane substrate) were held constant at 10% (v/v). Assays were then extracted twice with 1 mL of EtOAc and dried under a stream of nitrogen. The resultant residue from each assay was separately resuspended in 100 μL of acetonitrile (for assay containing baccatin III) or methanol (for assays containing 10-DAB (5)) and analyzed by LC-ESI-MS/MS. To calculate kinetic constants, each substrate was varied (1− 3000 μM) in separate assays under predetermined steady-state conditions. Assay products were quantified as described above. Kinetic parameters (KM and kcat) were calculated by nonlinear regression with Origin 9.0 using the following equation v0 = kcat [Eo][S]/(KM + [S]) (Northampton, MA) (Figures S16−S19). Although KM is not a true dissociation constant, in this study, it will serve as a means of comparing enzyme interactions with both substrates. Assays were prepared as follows: the taxane was added to a 5 mL glass test tube, and 200 mM MOPS (pH 8) was added dropwise to facilitate mixing of the acetonitrile or methanol. This mixture was incubated at 31 °C for 10 min before the acyl CoA and MBP-BAPT (0.2 μg, 0.024 nmol, 0.12 μM) were added to start the reaction. Liquid Chromatography Mass Spectrometry: BAPT Assay Analysis. An autosampler (at 10 °C) connected to a UPLC system (Waters Corp., Milford, MA) injected 10-μL of each processed assay onto an Ascentis Express C18 HPLC column (2.7 μm, 5 cm × 2.1 mm, at 30 °C, Sigma-Aldrich). The reaction products that were dissolved in acetonitrile or methanol were analyzed with a UPLC method using acetonitrile or methanol as the mobile phase, respectively. The column was eluted at 0.4 mL/min with 2.5% solvent B (100% methanol) and 97.5% solvent A (0.5% formic acid in water) with a 0.5 min hold, an immediate increase to 30% solvent B, followed by a linear gradient to 90% solvent B over 4 min, then increased to 100% solvent B over 0.5 min, and finally lowered to 2.5% solvent B over 0.5 min. The needle was washed with 0.4 mL each of 100% isopropanol and then with 10% acetonitrile in water prior to each injection. The HPLC effluent was directed to an electrospray ionization tandem mass spectrometer (Quattro Micro, Waters Corp, Milford, MA) in positive ion mode, with a cone voltage of 20 V and collision

an additional 2 h. The reaction was stopped by acidification (pH 5) with 8.8% formic acid, followed by ethyl acetate (2 mL) addition. Docetaxel (6) was added to the reaction as an internal standard (1 μM) and extracted twice with EtOAc (2 × 2 mL). The organic layers were pooled and dried under a nitrogen stream. Coupled assay products were analyzed by LC-ESI-MS/ MS with multiple reaction monitoring of the N-DBz2DP (8) transition ion [M + H]+ = m/z 734 → m/z 509 and of the internal standard (docetaxel) (1 μM) transition ion [M + H]+ = m/z 808 → m/z 509. Product peaks were quantified by calculating the ratio of the peak area for the transition ion abundance of 8 to that of the internal standard 6 at 1 μM. The amount of product formed was converted to units of activity (μmol/min). Total protein was quantified by Coomassie Bradford assays. Enzymatic Production of PheIso CoA (10). A large-scale preparative PheAT enzymatic assay was performed under the following conditions. A concentrated solution of PheAT (0.52 mM, 720 mg in 20 mL) in 10 mM HEPES buffer (pH 8) was stirred at 23 °C. MgCl2·(6H2O) (100 mg) and PheIso (9) (0.17 mmol) were dissolved in the PheAT solution. Separately, ATP (0.17 mmol) and CoA (0.13 mmol) were dissolved in 2 mL each of 10 mM HEPES, pH 8. Solutions of ATP and CoA were further adjusted to pH 8 (0.5 M NaOH), if necessary, before adding them to the stirred PheAT solution. Reaction progress was monitored at A258 by UV-HPLC to follow the (PheIso CoA) (10) produced and CoA that remained (Figure S6). After 7.5 h, 50% of the CoA was converted to thioester product, and the reaction was stopped by addition of 8.8% formic acid to pH 4 to precipitate PheAT and stabilize thioester 10. The precipitated reaction was centrifuged at 5000g for 10 min. The supernatant was collected, and the pellet was washed with water (pH 4 with formic acid) and recentrifuged. Supernatants were combined and filtered through a Millipore Amicon Ultra 30 kDa concentrator to remove trace protein. The flow-through was collected, flash-frozen in liquid nitrogen, and lyophilized. The lyophilized crude PheIso CoA (10) product was dissolved in 2 mL of 0.1% trifluoroacetic acid in distilled water (pH 4) and filtered for preparative purification on an HPLC (Agilent 1100 Series). Approximately 20 aliquots (100 μL) of the solution were loaded onto a preparative C18 column (Atlantis C18 OBD, 5 μm, 19 mm × 150 mm), with A258 monitoring of the effluent. The column was eluted at 4 mL/ min with 2.5% solvent B (100% acetonitrile) and 97.5% solvent A (0.1% trifluoroacetic acid in water) with a 4 min hold, a linear gradient to 30% solvent B over 15 min, then increased to 100% solvent B over 2 min, and finally lowered to 2.5% solvent B over 2 min. Fractions eluting at the retention time corresponding to the thioester product were collected, flashfrozen, and lyophilized to yield (2R,3S)-phenylisoserinyl CoA (97.5% pure, 45.5 mg, 0.05 mmol) at 30% yield relative to (2R,3S)-phenylisoserine. The purified thioester product was analyzed by NMR, UV-HPLC, mass spectrometry (Figures S7−S10) and the Ellman’s assay for free thiol (see Supporting Information). 1H NMR (500 MHz, D2O, pH 3 with CD3COOD) δ: 0.59 (s, 11″), 0.73 (s, 10″), 2.16 (br s, 6″), 2.72 (br s, 9″), 2.95−3.30 (m, 5″, 8″), 3.37 (m, 5′), 3.64 (m, 5′), 3.85 (s, 3″), 4.02 (br s, 1″), 4.38 (d, J = 7.32 Hz, 2c), 4.46 (d, J = 6.35 Hz, 4′), 4.74 (m, 2′, 3′, 3c), 4.89 (br s, NH2), 5.99 (d, J = 5.86 Hz, 1′), 7.20 (br s, aromatic H), 8.19 (s, adenine H), 8.44 (s, adenine H). (See Figure S8 for proton numbering) 13 C NMR (126 MHz, D2O) δ: 20.48 (s, 11′), 23.17 (s, 10′), D

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In vitro Biocatalysis of N-2-FP (14) in a Cascade Reaction. Four enzymes (PheAT, BAPT, BadA, and NDTNBT) were incubated together to produce N-2-FP (14) from the precursor baccatin III. The ligase reactions containing a mixture of PheAT and BadA were prepared on ice and incubated in a water bath (31 °C) for 5 min prior to the addition of the ligases. Baccatin III (0.5 mM dissolved in methanol) was added to the bottom of a glass test tube and mixed with 200 mM MOPS, pH 8. Substrates were then added to final concentrations of 3.5 mM MgCl2, 1 mM ATP, 1 mM CoA, 1 mM (2R,3S)-PheIso, and 1 mM 2-furoic acid. BadA (0.25 μg, ∼ 0.003 nmol) and PheAT (∼0.25 mg, 4 nmol) were then added to start the assay after temperature equilibration. The assay also contained 10% methanol (v/v) to ensure solubility of taxanes during the reaction in 200 μL total. MBPBAPT (14 μg, 0.15 nmol, 0.6 μM) and NDTNBT (91 μg, 1.7 nmol, 6.8 μM) were added to a final volume of 250 μL and incubated for 3 h. Docetaxel (1 μM) was added as an internal standard, and the solution was extracted with ethyl acetate (2 × 1 mL). The organic fraction was removed, dried under a stream of nitrogen, and resuspended in 100 μL of methanol. The reaction was analyzed for N-DBzP (12) and N-2-FP (14) by LC-ESI-MS/MS and multiple reaction monitoring of the transition ions ([M + H]+ = m/z 750 → m/z 509) (Figure S20) and [M + H]+ = m/z 844 → 509 (Figure S21), respectively. Product concentrations were normalized relative to the internal standard of docetaxel (1 μM).

energy of 16 eV. Each taxane was quantified by multiple reaction monitoring of the [M + H]+ → m/z 509 transition, common to both baccatin III, 10-DAB, and docetaxel. Peak areas were calculated using the MassLynx data analysis software, (Waters Corp., Milford, MA) and then converted to product concentrations using the docetaxel internal standard of 1 μM. In Vitro Biocatalysis of N-DBz2DP (8) in a Two-Enzyme Cascade Reaction. Baccatin III (0.75 mM or 3 mM dissolved in methanol) was added to several 5 mL glass tubes, followed by 200 mM MOPS (pH 8), 3.5 mM MgCl2, 1 mM ATP, and 1 mM PheIso. Varying amounts of CoA (0.05, 0.1, 0.25, 0.5, 1, and 2 mM) were added to separate tubes, followed by PheAT (0.75 mg/mL) to a final volume of 200 μL with 10% (v/v) methanol. The reaction was incubated for 1 h at 31 °C, and then MBP-BAPT (2 μg, 0.0024 nmol, 0.012 μM) was added and incubated for an additional 2 h. Docetaxel (13) (1 μM) was added as an internal standard and the solution was extracted with ethyl acetate (2 × 1 mL). The organic fraction was removed, dried under a stream of nitrogen, and resuspended in 100 μL of methanol. The reaction was analyzed for N-DBzP (12) by LC-ESI-MS/MS and multiple reaction monitoring of the transition ion [M + H]+ = m/z 750 → m/z 509 (Figure S20). Product concentrations were normalized relative to the internal standard of docetaxel (1 μM) ion [M + H]+ m/z 808 → m/z 509 transition. BadA Kinetic Parameters with 2-Furoic Acid (15). Stocks of ATP (1 mM) and CoA (1 mM) were dissolved in Assay Buffer (50 mM NaPO4 containing 5% glycerol, pH 8.0), MgCl2 (5 mM) was stored in water, and stocks of 2-furoic acid (15) (0.1−100 mM) were dissolved in methanol. Note, methanol concentrations were held at 1% (v/v) among assays containing different concentrations of 15. We established steady-state kinetic rates of BadA (heterologous enzyme expression is described in a previous report43) with respect to protein concentration, time, and additional reagents. ATP (250 μM), CoA (250 μM), and MgCl2 (750 μM) were dissolved in Assay Buffer and preincubated at 31 °C for 10 min, and then BadA was added (0.3 μg/mL; in 90 μL total volume). The reaction was quenched (pH 3) with 10% formic acid in water. Acetyl CoA (1 μM final concentration) was added as an internal standard. An autosampler (at 10 °C) connected to a UPLC system (Waters Corp., Milford, MA) injected a 10-μL aliquot of each assay onto an Ascentis Express C18 HPLC column (2.7 μm, 5 cm × 2.1 mm, at 30 °C, Sigma-Aldrich). The column was eluted at 0.4 mL/min with 2.5% solvent B (100% acetonitrile) and 97.5% solvent A [0.05% triethylamine in water (pH 5.0− 6.0)] with a 0.5 min hold, followed by a linear gradient to 20% solvent B over 4 min, then increased to 100% solvent B over 2 min, and finally lowered to 2.5% solvent B over 0.5 min. The UPLC effluent was directed to an electrospray ionization mass spectrometer (Quattro Premier XL, Waters Corp., Milford, MA) in negative ion mode, with a cone voltage of 60 V and collision energy of 44 eV. 2-Furoyl CoA (16) was quantified by multiple reaction monitoring of the [M − H]− m/z 860 → m/z 408 transition, common to acyl CoA thioesters.42,47 Peak areas (calculated using the MassLynx data analysis software, Waters Corp., Milford, MA) were converted to product concentrations using a standard curve for a series of benzoyl CoA concentrations (100 nM to 15 μM) that were normalized to an internal standard acetyl CoA (1 μM).

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RESULTS AND DISCUSSION A Taxus plant phenylpropanoyltransferase, BAPT, transfers the phenylpropanoyl side chains of PheIso CoA (10) and β-Phe CoA (3) to the taxane baccatin III (6), a paclitaxel precursor (see Figure 2).34 Characterization of BAPT kinetic parameters is difficult since the amino acyl-CoA substrates used here are not commercially available. Therefore, the substrates PheIso CoA and β-Phe CoA were biosynthesized (using the enzyme PheAT) and synthesized, respectively. PheAT was derived from a full-length initiation module of the nonribosomal peptide synthetase (NRPS) that encodes the adenylation, thiolation, and epimerization domains (PheATE is the full-length) on the pathway to the cyclic decapeptide tyrocidine A.48 The PheA portion of PheATE ordinarily uses ATP and Mg2+ to activate (S)-α-phenylalanine to a phosphoric acid-AMP anhydride. The phenylalanyl moiety is then transferred to the mercapto group of 4′-phosphopantetheine of the bordering thiolation (T) domain, with release of AMP. The epimerization (E) domain then changes the stereochemistry of the pendent (S)-α-Phe to the (R)-enantiomer, which progresses through synthetase modules TycB and TycC to produce tyrocidines A−D in Bacillus brevis.49 PheAT used in this work was modified to remove the epimerization domain, leaving the adenylation and thiolation domains to work as a CoA ligase. An earlier study showed that PheAT could convert PheIso (and several other arylisoserines) to their CoA thioesters.42,47 Isoserinyl CoA thioesters are biosynthetic precursors on the pathway to analogues of paclitaxel (1), which are cytotoxic against multiple drug-resistant (MDR) cancer cell lines or are in clinical trials.35−41 Therefore, we used ∼1 g of PheAT to biocatalyze PheIso CoA (10) in preparative-scale yield. The scaled biocatalytic production of PheIso CoA allowed us to calculate the kinetic constants of the BAPT acyltransferase and compare those with values estimated in an earlier study.34 βE

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Biochemistry Table 1. Kinetic Constants of Enzymes Needed to Biocatalyze N-2-FP (14) from Baccatin IIIa substrate 1 KMb (μM) Entry 1 2 3 4 5 6

Acyltransferase MBP-BAPT β-Phe CoA (3) (5.6 ± 1.0) MBP-BAPT PheIso CoA (10) (22 ± 7) NDTNBT benzoyl CoA (375 ± 67) NDTNBT benzoyl CoA (410) Ligase PheAT PheIso (9) (440 ± 62) BadA 2-Furoic acid (15) (410 ± 0.65)

substrate 2 KMb (μM)

kcat (s−1)

kcat/KMc (M−1 s−1)

kcat/KMd (M−1 s−1)

ref

baccatin III (6) (68 ± 9.6) baccatin III (6) (see above) N-DBzP (12) (78 ± 11) N-DBz2DP (8) (450)

0.058 ± 0.001 0.002 ± < 0.001 ∼0.6 ± > 0.01 N/D

10,000 91 1,600 N/A

850 29 7,700 N/A

this work this work 36 39

CoA (210 ± 57) CoA (4.4 ± 0.65)43

0.015 ± 0.0028 0.6 ± 0.04

34 150

72 14,000

42 this work

a

Abbreviations: Enzyme abbreviations are in the main text; ND: not determined and N/A: not applicable. bKM of the enzyme for the substrate listed was measured with the cosubstrates at apparent saturation. cUsing KM for Substrate 1. dUsing KM for Substrate 2.

Figure 3. Various characterized β-amino phenylpropanoid taxanes from Taxus plants obtained from a putative 5-O-deacyl precursor through route i to (left) 5-O-β-Phe taxa-11(12),4(20)-dienes and route ii to (center) 5-O-PheIso taxa-11(12),4(20)-dienes, and from a putative 13-O-deacyl precursor through route iii to (right) 13-O-PheIso taxa-11(12)-en-4(5)-oxetane compounds.

Phe CoA (3) was synthesized by a previous method34 to compare its turnover with that of PheIso CoA. Synthesis of Amino Acyl-CoA Substrates. Biocatalysis of PheIso CoA (10). The synthesis of PheIso CoA (10) in an earlier study was low-yielding because of poor yielding steps and multiple protecting group manipulations.34 This prompted us to employ PheAT to make the PheIso CoA thioester. We estimated from the kinetic parameters of PheAT(kcat ≈ 0.015 s−1 and KM ≈ 440 μM for PheIso (9), and KM ≈ 208 μM for CoA) that ∼700 mg of enzyme would theoretically produce ∼100 mg of PheIso CoA in 1 h (Table 1).42,47 PheAT (∼1 g) was heterologously expressed and purified (to >90%) as a 6× His-tagged fusion protein from E. coli. The PheAT-catalyzed reaction progress was monitored by UV-HPLC over 19 h. The converted yield of PheIso CoA (10) reached ∼50% relative to CoA. The reaction was stopped with formic acid, and the PheIso CoA (46 mg, 50 μmol, 30% conversion relative to PheIso) was purified to 97% purity, which was verified by preparative reverse-phase HPLC, NMR, and the Ellman’s test.50 The preparative-scale biocatalysis of PheIso CoA described here is a significant improvement over the earlier synthetic method that produced only ∼2.7 mg of the thioester (2.9 μmol, ∼80% purity) in ∼10% yield relative to PheIso.34 Synthesis of (3R)-β-Phe CoA. β-Phe CoA (3) was synthesized by a mixed anhydride method described in an earlier procedure.34 Briefly, β-Phe was N-Boc protected and then converted to a mixed anhydride intermediate. The

anhydride was converted to a thioester after reaction with CoA, and the amino acyl CoA was N-deprotected with trifluoroacetic acid to make the β-Phe CoA (82% yield relative to CoA; 95% purity). Kinetics of BAPT for β-Phe CoA, PheIso CoA, and Baccatin III. Earlier activity assays used recombinant BAPT expressed without an affinity epitope-tag for purification.39 Thus, only crude preparations of BAPT expressed in E. coli were available, and the soluble enzyme fraction was estimated to contain ∼1% of the total soluble bacterial protein in the previous study.39 Further, the earlier study was confounded by limited quantities of synthetically derived PheIso CoA (10) and radiolabeled [13-3H]baccatin III substrates. These limitations prevented complete characterization of BAPT with these substrates to produce N-DBzP (12). Therefore, only estimated KM values for baccatin III (2.4 μM) and the β-Phe CoA thioester (3) (4.9 μM), not the kcat, were reported for making the N-DBz2DP (8) product. In this study, we expressed BAPT as a maltose binding protein fusion (MBP-BAPT) to increase its solubility and attached a C-terminal His6-epitope affinity tag. The expressed MBP-BAPT was purified to >70% and isolated at 300 μg/mL in ∼2 mL. A combination of (1) purifying and quantifying overexpressed MBP-BAPT in soluble form and (2) making sufficient amounts of phenylpropanoyl CoA substrates enabled us to calculate the kcat values of BAPT for baccatin III (6), βPhe CoA (3), and PheIso CoA (10) for the first time (Table 1). This information allowed us to begin constructing an F

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Figure 4. (Left) N-Debenzoylpaclitaxel catalyzed from PheAT and BAPT sequential reactions. Production of N-DBzP at two different concentrations of baccatin III at 0.75 mM (open circles, ○), 3 mM (black squares, ■), (n = 2). Outlier data points deviating from the Michaelis− Menten curve fit are encircled (open circles) or boxed (black squares). (Right) Representative reverse-phase LC-ESI/MS/MS analysis in selected ion mode of [M + H]+ = m/z 750 ion for N-DBzP and the [M + H]+ = m/z 808 ion for the internal standard docetaxel.

plants does not support a principal pathway to paclitaxel that proceeds through a C13-(β-phenylalanyl)baccatin III (i.e., NDBzDP (8) intermediate despite the higher catalytic efficiency of BAPT for the β-Phe CoA (3) substrate. This suggests a pathway on which only a PheIso group is transferred to the C13 hydroxyl of oxetane taxanes in Taxus plants. Intrinsic Kinetics of MBP-BAPT for PheIso CoA and 10Deacetylbaccatin III. MBP-BAPT catalysis also produced Nde-(t-butoxycarbonyl)-2′-deoxydocetaxel (7), a precursor of the drug docetaxel, using PheIso CoA (10) and 10-deacetylbaccatin III (10-DAB) (5) as cosubstrates. The parameters kcat and KM of MBP-BAPT for PheIso CoA and 10-DAB (at saturation) could not be calculated because the activity was reduced ∼90fold for 10-DAB compared with that for baccatin III. Instead, vrel values are reported (Figure S19). With cosubstrates at apparent saturation, BAPT turned over 10-DAB and PheIso CoA to the target product 7 at a vrel of 1.92 × 10−4 nmol/min compared to a turnover of baccatin III and PheIso CoA at 0.018 nmol/min for the same enzyme stock. These data suggest that the 10-acetyl group of baccatin III facilitates substrate turnover by MBP-BAPT and supports that baccatin III is likely the natural substrate of BAPT on the paclitaxel (1) pathway in Taxus plants. Coupled PheAT and BAPT Catalysis to Biocatalyze NDebenzoylpaclitaxel. The feasibility of combining PheAT and MBP-BAPT in a one-pot coupled reaction to biocatalyze NDBzP (12) was evaluated with a series of in vitro assays. The PheAT ligase and phenylpropanoyltransferase MBP-BAPT were incubated with baccatin III (6), PheIso and varied amounts (50 μM to 2 mM) of CoA to determine optimal NDBzP biocatalysis conditions (Figure 4). N-DBzP was produced maximally ≥7.5 nmol (37.5 μM, 5.7 μg in 200 μL) in the coupled reaction when baccatin III was above 0.75 mM (up to 3 mM), and CoA was at 1 mM (∼5 times the KM of PheAT for CoA) (Table 1) (Figure 4). These data also show that a high concentration (2 mM) of CoA reduced N-DBzP production, likely through competitive inhibition with the acyl CoA active site in MBP-BAPT and possible feedback inhibition with PheAT. In Vitro Cascade Reaction to Biocatalyze N-2-FP (14). Four enzymes were used in vitro in one pot to produce Ndebenzoyl-N-(2-furoyl)paclitaxel (N-2-FP) (14) from the precursor baccatin III (6) (Figure 2). Previously reported kinetic constants of PheAT,42 NDTNBT,35 as well as those

enzyme cascade reaction using BAPT to make a biologically active paclitaxel analogue. The KM of purified MBP-BAPT for baccatin III (6) is 28-fold higher than that of wild-type BAPT for the same substrate used in an earlier study.39 By comparison, the KM of MBP-BAPT for β-Phe CoA (3) calculated here (Table 1) is similar to that of wild-type BAPT for β-Phe CoA (4.9 μM) reported in the earlier study.34 We note, the wild-type BAPT expressed earlier39 and its MBP-fusion protein used here are architecturally different, which likely contributed to their different KM values for the same substrate. The kcat of MBP-BAPT for PheIso CoA (10) was calculated in this study to be 27-fold slower, and its KM is 4-fold higher than for β-Phe CoA (3) (Table 1). However, since the 2′-hydroxyl group of the PheIso moiety in paclitaxel (1) and its variants is needed for bioactivity, calculating the kinetic parameters of MBP-BAPT with PheIso CoA was more important for constructing the biocatalytic cascade reaction toward a bioactive 2-furoyl paclitaxel analogue described herein. The higher catalytic efficiency of MBP-BAPT for transferring the β-phenylalanyl moiety from β-Phe CoA (3) (kcat/KM ≈ 104 M−1 s−1) compared with transferring the isoserinyl moiety from PheIso CoA (10) (kcat/KM ≈ 90 M−1 s−1) suggests that the proposed paclitaxel biosynthetic pathway may indeed advance through an N-DBzDP (12) intermediate (see Figure 2). Likely, the hydroxyl group on the phenylpropanyl side chain of PheIso CoA creates a steric and/or electronic barrier within the active site that affects the turnover rate. It should be noted that several different, yet low abundance taxa-11(12),4(20)-dienes (∼0.05% isolated yield from Taxus plants) have a β-Phe group attached at C5 of the taxane core (Figure 3, left), and several of their cognate dienes (∼0.04% isolated yield) have a PheIso moiety at C5 (Figure 3, center).16 The latter could suggest a biological progression where C5-βPhe taxa-11(12),4(20)-dienes are converted to their C5-PheIso derivatives by 2′-hydroxylation. Alternatively, the β-Phe and PheIso moieties present at C5 of these taxadienes could indicate that the side chains are transferred independently from their respective CoA thioesters. By contrast, 31 taxanes with a PheIso moiety at C13 (∼0.11% isolated yield from Taxus plants) belong to the oxetane taxane family that includes paclitaxel (1) (Figure 3, right).16 To date, no naturally occurring taxanes with a β-phenylalanyl side chain at C13 have been identified. Thus, metabolite occurrence in Taxus G

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Figure 5. (A) LC-ESI-MRM trace in positive mode for the m/z 844 → 509 ion transition of the products made after incubation with PheAT, BAPT, BadA, and NDTNBT in the enzyme cascade reaction. Front profile (solid line): sample analysis of an assay that contained all substrates, cofactors, and enzymes; back profile (dotted line): sample analysis of an assay that contained all substrates, cofactors, and enzymes except NDTNBT. (B) The compound eluting at 4.2 min in panel A was fragmented with selection for the pseudo molecular ion [M + H]+ (m/z 844) in an MS/MS method. Diagnostic ions consistent with N-2-FP (MW 843) are shown; the [M + H]+ was below the detection limits.

calculated in this study for BadA for 2-furoic acid (Table 1, Figure S22) and MBP-BAPT were used to inform the design of the reaction cascade. An additional goal is to provide exogenous cosubstrates at saturation for each enzyme in order to drive the forward reaction.51 We used the kinetic constants for each enzyme to calculate a theoretical maximum concentration of each intermediate made in the four-enzyme reaction. On the basis of these data, PheAT and BadA were projected to turnover PheIso (9) and 2-furoic acid (15), respectively, and CoA to 54 μM of PheIso CoA (10) and 45 μM of 2-furoyl CoA (16), prior to adding MBP-BAPT and NDTNBT. Despite the concentration of PheIso CoA being slightly higher (only 2.5-fold) than the KM of its downstream catalyst MBP-BAPT (Table 1), we knew the reaction would proceed based on previous results from the two-enzyme cascade under the same reaction conditions (Figure 4). By contrast, the 2furoyl CoA concentration was 10-fold lower than the KM of its downstream catalyst NDTNBT (Table 1) resulting in suboptimal flux conditions. The concentrations of reaction intermediates in the assay could not be monitored in real time in order to evaluate when steady-state flux was reached for each step. Therefore, we were unable to know when enzyme or substrate amounts needed to be adjusted to maintain steady state through the pathway. Further, blindly adding more BadA, CoA, or NDTNBT to try and drive the downstream reactions forward would also be challenging, since BadA, with its superior catalytic properties over PheAT (Table 1), would outcompete PheAT for CoA. Adding more CoA to overcome this barrier was projected to negatively affect BAPT turnover as observed previously in this study (Figure 4). In addition, since the expected concentration of 2-furoyl CoA (16) was suboptimal for NDTNBT, adding more of the enzyme would not enhance turnover. Despite these concerns, the one-pot, four-enzyme

cascade was successful. PheAT and BadA assays were run under saturating substrate concentrations to ensure maximum acyl CoA biosynthesis.52,53 The acyltransferases MBP-BAPT and NDTNBT were subsequently added (after 1 h) to complete the cascade reaction. As in the two-enzyme cascade reaction done earlier in this study, the PheAT and MBP-BAPT coupled reaction was projected to make ∼40 μM of N-DBzP (12), which is 2-fold lower than the KM of NDTNBT (Table 1). The calculated assay concentration of 2-furoyl CoA (∼45 μM) made by BadA is 10-fold lower than the KM (∼400 μM, estimated as similar to that for benzoyl CoA)35 of its downstream catalyst NDTNBT. The success of the enzyme cascade reaction relied in part on the substrate selectivity of NDTNBT for 2-furoyl CoA (16) made from BadA catalysis (Table 1).33,34,37 The shortcoming of NDTNBT is that the anticipated concentrations of its two substrates, fed by separate upstream biocatalytic pathways, are below the corresponding KM values of NDTNBT for the substrates. Therefore, the reaction was not ideally pseudo-firstorder for either substrate, and NDTNBT catalysis was considered to be suboptimal. Product Identification and Quantification of the in Vitro Cascade Reaction. We quantified the compounds in the product mixture from the cascade reaction by LC-ESI-MS/MS analysis with multiple reaction monitoring, using docetaxel (13) as an internal standard. The fragment ion profile showed a peak corresponding to an analyte with an m/z 844 → 509 transition at 4.2 min (Figure 5A). This ion transition was below the limits of detection in a control reaction lacking only NDTNBT. Fragmentation of the [M + H]+ = m/z 844 ion generated daughter ions that were consistent with those of N-2FP (14) (Figure 5B).54,55 The most diagnostic ions were m/z 276, consistent with a protonated N-(2-furoyl)phenylisoserine H

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Biochemistry acid [2-FurPheIso + H]+, and m/z 230, consistent with a side chain fragment ([2-FurPheIso + H]+ − H2O − CO). These ion fragments supported the formation of the N-2-furoyl derivative of paclitaxel (Figure 5B). The target product N-2-FP (14) (1.1 ± 0.1 μM, 230 ng, 0.30 nmol) was made at a 25-fold lower amount than the N-DBzP (12) intermediate (25 ± 9 μM, 5 μg, 6 nmol), which was consistent with earlier production levels observed in the twoenzyme cascade reaction containing PheAT and MBP-BAPT. This low product turnover was likely caused by the rate-limiting N-acylation step catalyzed by NDTNBT in this assay. The design of the cascade reaction intended to maximize flux by keeping biosynthetic intermediates above the KM of the downstream catalyst with exogenous cosubstrates at saturation. NDTNBT was fed by two biosynthetically derived cosubstrates whose concentrations were below the KM for the catalyst, yet N-2-FP (14) was indeed measurably produced. The production of 14 may have been boosted by CoA recycling after the acyltransferases released CoA, thus enabling the concentrations of the thioesters to increase more than expected. Conclusions. The preparative-scale biocatalysis of PheIso CoA (10) made it possible to characterize the intrinsic kinetic parameters of the phenylpropanoyl CoA transferase (BAPT) for the first time. A cascade enzyme reaction containing two bacterial ATP-dependent CoA ligases, PheAT and BadA, and the Taxus acyltransferases, BAPT and NDTNBT, produced the biologically active, anticancer analogue N-2-FP (14). The latter is one example of several analogues that can be made through this cascade reaction. More importantly, N-(2-furoyl) paclitaxel analogues presented in earlier studies were shown to be 3.5−6fold more cytotoxic (IC50) than paclitaxel (1) against J774.1 mouse macrophage cells.45,56 The N-DBzP precursor (12) of N-2-FP (14), made biocatalytically in this study, can be Nacylated, either synthetically57 or biochemically35 to various efficacious paclitaxel analogues. Coupling PheAT and BAPT reactivity also converted 10-DAB (5) and PheIso CoA (10) to N-de-(t-butoxycarbonyl)docetaxel (N-DBocD) (11) at a slower rate. This common precursor is of commercial interest toward in vitro or in vivo biocatalysis of the anticancer drug docetaxel, which is used in combination-drug therapy against triple negative breast cancer.58−60 Continued efforts to improve the enzyme cascade reaction described here will require further fine-tuning of each enzyme amount so intermediates reach steady-state concentrations to maximize flux to the products. The cascade reaction used in the present study begins with advanced pathway precursors baccatin III (6) or 10-DAB (5). These compounds are available at ∼4-fold the levels of paclitaxel (1) isolated from Taxus plant cell fermentation bioreactors or Taxus plant leaves.4,61 Incorporating a baccatin III precursor into a streamlined reaction cascade to make target paclitaxel analogues reduces side reactions and thus simplifies the purification of the target product from hundreds of chemically similar compounds, such as those found in Taxus cells. Employing four soluble enzymes in a tandem in vitro reaction cascade to make taxane drugs reduces the need to reverse engineer several eukaryotic plant enzymes such as membrane-bound P450 hydroxylases25 to function in prokaryotic hosts to complete the ∼19-step paclitaxel pathway in vivo.26,62,63 Future research will focus on optimizing the four-enzyme cascade reaction for in vivo expression and activity to make analogues of paclitaxel (1). An earlier feeding study showed evidence that E. coli engineered to express a 10-O-

acetyltransferase (DBAT) could uptake an advanced taxane structure, 10-DAB (5), and acylate it to baccatin III (6) in vivo.64 Thus, it becomes feasible that a baccatin III precursor can be fed to E. coli to make paclitaxel compounds in vivo. Continued development of bioengineered systems will necessarily include tuning protein expression levels and fitness, substrate concentrations, and growth conditions.65,66 Expansion of substrate specificity within the four-enzyme cascade reaction scheme, described herein, can be facilitated by rational active site mutagenesis.65 This has already been demonstrated with BadA for the development of expanded, novel activities from single-point mutants.43 Previously, a homology model of PheAT guided mutagenesis efforts to expand its substrate specificity to include isoserinyl CoA substrates of MBP-BAPT.42 Further expanding the substrate specificity of PheAT for variously substituted isoserinyl CoA thioesters will also aid in analogue diversification. An earlier study showed the complexity of synthesizing an isoserinyl CoA,39 and PheAT catalysis42 was used here to biocatalyze an amino acid thioester (PheIso CoA (10)) at preparative scale. Thus, we envision that PheAT can biocatalyze a series of isoserinyl CoA thioesters to further test the substrate specificity of MBP-BAPT, which has only been tested with four synthetically derived amino phenylpropanoyl CoA substrates.34 As important, beyond just making paclitaxel analogues, isoserinyl CoA thioesters may be useful in other bioengineering applications to make new natural product analogues with novel bioactivities. Combinations of isoserinyl CoA thioesters and plant acyltransferases hold potential for regiospecific acylation toward novel biosynthetic products,67−70 compared to synthetic acylation reactions, which may lack regiospecificity and thus need additional protecting group steps.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00912. Experimental Methods, Instrumentation, Materials, Kinetics, and NMR data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kevin D. Walker: 0000-0001-5208-6692 Funding

The authors are grateful to the Michigan State University Office for Inclusion and Intercultural Initiatives for supporting the MSU Chemistry 4-Plus Bridge to the Doctorate Program (GA017081-CIEG), and to the Michigan State University AgBioResearch Grant RA078692−894. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank undergraduate Manisha Munasinghe for her technical assistance, and Professor Rodney B. Croteau and the Washington State University Research Foundation for the cDNA encoding the enzymes NDTNBT and BAPT. We also thank the Michigan State University Research Technology I

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Support Facility: Mass Spectrometry and Metabolomics Core for their technical assistance.

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