Characterization of FK506 Biosynthetic Intermediates Involved in Post

May 24, 2013 - Intelligent Synthetic Biology Center, Korea Advanced Institute of Science and ... that the post-PKS biosynthetic pathway of FK506 start...
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Characterization of FK506 Biosynthetic Intermediates Involved in Post-PKS Elaboration Yeon Hee Ban,†,‡ Pramod B. Shinde,†,‡,§ Jae-yeon Hwang,† Myoung-Chong Song,†,⊥ Dong Hwan Kim,∥ Si-Kyu Lim,∥ Jae Kyung Sohng,∇ and Yeo Joon Yoon*,†,§ †

Department of Chemistry and Nano Science and §Institute of Nano-Biotechnology, Ewha Womans University, Seoul 120-750, Republic of Korea ⊥ Intelligent Synthetic Biology Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea ∥ GenoTech Corporation, Daejeon 305-343, Republic of Korea ∇ Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Chungnam 336-708, Republic of Korea S Supporting Information *

ABSTRACT: The post-PKS modification steps of FK506 biosynthesis include C9-oxidation and 31-O-methylation, but the sequence of these reactions and the exact route have remained unclear. This study details the post-PKS modification pathways in FK506 biosynthesis through the identification of all intermediates and in vitro enzymatic reactions of the cytochrome P450 hydroxylase FkbD and the methyltransferase FkbM. These results complete our understanding of post-PKS modification steps to FK506 showing the substrate flexibility of two enzymes involved and the existence of two parallel biosynthetic routes to FK506.

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oxidation catalyzed by cytochrome P450 hydroxylase (FkbD) and 31-O-methylation by S-adenosylmethionine (SAM)dependent methyltransferase (FkbM).9,10 Although the entire biosynthetic gene cluster of FK506 and the previously undescribed allylmalonyl-CoA biosynthetic pathway have recently been characterized,8 the detailed post-PKS modification route has remained unresolved. During the review process of this article, a similar study regarding the post-PKS biosynthetic pathways of FK506 was published.11 However, the structural characterization of the biosynthetic intermediate was incomplete. In this study, we report a comprehensive characterization of all the FK506 biosynthetic intermediates involved in post-PKS modification based on in-depth NMR, HPLC−ESI-MS/MS, and high-resolution MS (HR-MS) data. It has been previously reported that FkbM catalyzes Omethylation at the C31-position of the macrolactone ring. The enzymatic function of FkbM was demonstrated by trans-

K506 (1, also known as tacrolimus) is a macrocyclic polyketide of microbial origin exhibiting various biological activities, including immunosuppressive,1 antifungal,2 antiinflammatory,3 neuroprotective, and neuroregenerative.4 It is a clinically important drug used to prevent the rejection of organ transplants and to treat autoimmune diseases such as atopic dermatitis.5 The immunosuppressive activity of FK506 results from its ability to inhibit interleukin 2-mediated T-cell proliferation by the interaction of FK506−FKBPs (FK506binding proteins) complex with calcineurin.6 FK506 (1) is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. The biosynthesis of FK506 is initiated by the incorporation of the chorismate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a starter unit,7 which in turn is elongated by 10 condensation steps with two malonyl-CoA, two methoxymalonyl-acyl carrier proteins, five methylmalonyl-CoA, and an allymalonyl-CoA.8 The linear polyketide chain is attached with a pipecolate by the NRPS FkbP; subsequent cyclization generates the macrolide ring.9 The final formation of FK506 requires further post-PKS modification steps, including C9© 2013 American Chemical Society and American Society of Pharmacognosy

Received: February 7, 2013 Published: May 24, 2013 1091

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Figure 1. Proposed post-PKS modification pathway for FK506 (1) biosynthesis.

Figure 2. HPLC−ESI-MS chromatograms obtained from the culture of ΔfkbDin‑frame strain (A), ΔfkbDapr strain (B), ΔfkbMapr strain (C), and partially purified fraction of the culture of Streptomyces sp. KCTC116048BP (D).

thylFK506 (3) due to a possible polar effect on the expression of the downstream gene f kbM.13,14 The accumulation of 3 also suggested that FkbM methyltransferase, which has a relatively stringent substrate specificity toward oxygenated intermediates, acts after the FkbD oxygenase. These previous results suggested that the post-PKS biosynthetic pathway of FK506 starts from 3,

formation of 31-O-demethylFK506 (2) into FK506 by the purified FkbM12 and the accumulation of 2 as a result of the inactivation of the f kbM gene.13,14 The evidence that FkbD is responsible for the formation of the C9-carbonyl group of FK506 was obtained by gene disruption. An inactivation of f kbD resulted in the accumulation of 9-deoxo-31-O-deme1092

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Table 1. 13C and 1H NMR Data of Compounds 2−4 in CDCl3 2 (major rotamer) position

δC

1 2 3

169.2 56.7 27.8

4

21.3

5

24.6

6

39.4

7 8 9

165.0 196.2b

10 11 12

97.2 34.8 32.8

13 14 15 16

73.8 73.0 75.0 35.4

17 18

26.2 48.8

19 20 21 22 23

139.1 122.7 53.0 212.5b 43.8

24 25 26 27 28 29 30

70.1 40.0 77.7 132.4 130.1 35.1 39.2

31 32 33

75.5 75.4 32.2

34

31.1

35

35.7

36 37 38 39 40 41 42 43 44 45 10-OH

135.7 116.8 16.4 20.6 16.1 9.7 14.1 56.5 57.2

δH, m (J in Hz) 4.58, 2.05, 1.90, 1.74, 1.38, 1.70, 1.42, 4.39, 3.00,

d (5) m m m m m m d (12.5) t (13)

2 (minor rotamer) δC 169.0 52.9 26.3 20.9 24.7 44.1

3 (major rotamer)

δH, m (J in Hz) 4.97, m 2.30, m 1.76, m 1.74, m 1.38, m 1.66, m 1.50, m 3.69, m 3.26 (overlapped)

166.0 192.9

2.30, m 2.12, m 1.50, m 3.36 (overlapped) 3.68, d (9.5) 3.55, m 1.58 (overlapped) 1.32, m 1.68, m 2.16, m 1.80, m 5.01, m 3.38 (overlapped) 2.75, 2.06, 3.90, 1.85, 5.30,

dd (16, 3) m m m br s

5.08, d (9) 2.32, m 1.87, m 1.14, m 3.55, m 3.56, m 1.94, m 1.34, m 1.60 (overlapped) 1.02, m 2.45, m 2.16, m 5.70, ddt (17, 10, 7) 5.00, br s 0.97, d (6.5) 0.91, d (6.5) 1.58, s 0.84, d (6.5) 1.60, s 3.37, s 3.28, s

98.7 33.7c 32.7 73.8 72.4 76.8 nad 26.2 48.5 139.8 122.9 50.8 na 44.1 69.1 40.5 78.0 132.0 129.7 33.6c 35.1 75.5 75.0 32.1 33.2 35.6 135.5 116.8 16.4 19.6 16.0 10.0 14.4 56.3 57.8

δC

a

169.6 52.8 26.8 20.9 24.6 42.8

174.3 37.5

2.25, 2.10, 1.48, 3.43, 3.84, 3.56, na

m m m m dd (10, 9) m

1.68, m 2.16, m 1.86, m

74.5 70.8 77.1 36.5 26.0 48.5 141.3 121.5 53.6 214.5b 42.5

5.01, m 3.44, m 2.65, 2.32, 3.92, 1.85, 5.17,

98.7 38.6 32.8

d (17) m m m br s

69.6 40.5 76.8 132.6 128.9 35.2 39.1

5.03, d (9.5) 2.20, m na 3.32 (overlapped) 3.40, m 1.94, m 1.34, m 1.50, m 1.02, m 2.45, m 2.19, m 5.70, ddt (17, 10, 7) 5.00, br s 0.94, d (6.5) 0.82, d (6.5) 1.61, s 0.88, d (7) 1.63, s 3.36, s 3.33, s

4.21, s

75.6 75.0 32.2 31.2 36.1 135.6 116.8 17.1 18.9 16.0 10.0 14.6 56.3 57.9

δH, m (J in Hz) 4.85, 2.23, 1.70, 1.70, 1.21, 1.68, 1.51, 3.69, 3.20,

d (4) m m m m m m d (10.5) td (12.5, 3)

2.67, d (15.5) 2.49, d (15.5) 1.54, m 1.96, m 1.52, m 3.42, m 3.85, dd (10, 9.5) 3.52, m 1.48, m 1.33, m 1.68, m 2.30, m 1.63 (overlapped) 5.10, d (8.5) 3.31, m 2.63, 2.23, 3.99, 1.85, 5.18,

d (17) m dd (9.5, 4) m br s

4.96, 2.30, 1.87, 1.11, 3.31, 3.41, 1.96, 1.32, 1.60, 1.04, 2.40, 2.21, 5.71, 5.00, 0.94, 0.76, 1.65, 0.86, 1.66, 3.36, 3.36,

d (9.5) m m m m m m m m m m m ddt (17, 10, 7) br s d (6) d (6) s d (6.5) s s s

7.06, s

1093

4 (major rotamer) δC

a

169.6 52.8 26.8 20.9 24.6 42.8

174.2 37.5 98.7 38.7 32.8 74.5 70.8 77.1 36.4 25.9 48.6 141.4 121.5 53.6 214.8b 42.5 69.5 40.6 76.8 132.6 129.0 35.1 35.1 84.4 73.7 31.4 30.9 36.1 135.6 116.8 17.1 18.9 15.9 10.0 14.7 56.3 57.9 56.8

δH, m (J in Hz) 4.85, d (5) 2.23, m 1.70, m 1.70, m 1.21, m 1.68 (overlapped) 1.51, m 3.69, d (11) 3.19, td (12.5, 2.5)

2.67, d (15.5) 2.49, d (15) 1.58, m 1.97, m 1.53, m 3.40 (overlapped) 3.85, d (9.5) 3.51, m 1.48, m 1.33, m 1.65 (overlapped) 2.33, d (12.5) 1.67 (overlapped) 5.10, d (8) 3.32 (m) 2.63, 2.23, 3.99, 1.86, 5.19,

d (18) m dd (10, 3.5) m br s

4.96, d (9.5) 2.28, m 2.02, m 0.96 (overlapped) 2.99, m 3.40 (overlapped) 1.98, m 1.35, m 1.62, m 1.04, m 2.42, m (7) 2.28, m 5.71, ddt (17, 10, 7) 5.00, br s 0.93, d (6) 0.76, d (6) 1.65, s 0.87, d (7.5) 1.67, s 3.36, s 3.35, s 3.39, s 7.06, s

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Table 1. continued a

Signals from the minor rotamer were also observed in the 13C NMR spectrum. bSignals were assigned from HMBC spectra. cSignals could be interchanged. dna = signals could not be assigned.

the known intermediate, 9-deoxo-31-O-demethylFK506 (3), due to the polar effect on the expression of the downstream gene f kbM, as previously reported.14 During HPLC−ESI-MS/ MS analysis of the ΔfkbDapr strain, the peak eluting at 16 min showed an ammonium adduct ion at m/z 793.1 (Figure 2B), and fragment ions at m/z 776.1, 758.0, 740.0, 722.0, and 561.9, which were identical to those obtained from a previous experiment (Supporting Information, Figure S12).14 The presence of the characteristic ion at m/z 561.9 derived from the C1−C24 fragment of 3 (m/z 576.0 for the C1−C24 fragment of FK506) indicated the loss of a C9-keto group.15 In addition, the difference of 28 Da between the ammonium adduct of 3 and that of FK506 (1, m/z 821.3; 3, m/z 793.1) could be accounted for by the loss of a C9-keto group as well as by the loss of a methyl group from the cyclohexane moiety. The f kbM inactivation mutant (ΔfkbMapr strain) generated through gene replacement with an apramycin resistance cassette using the PCR-targeting method led to the production of a known intermediate, 31-O-demethyl-FK506 (2).14 The HPLC−ESIMS/MS data of the ΔfkbMapr strain exhibited a peak at 14 min (Figure 2C), with its ammonium adduct ion at m/z 807.1, which was 14 Da less than that of FK506 ([M + NH4]+ at m/z 821.3), indicating the possibility of the loss of a methyl group. Moreover, its product ion spectrum contained fragment ions at m/z 790.1, 772.1, 754.0, 736.1, and 576.0. Considering the fragment ion at m/z 576.0 resulting from the C1−C24 fragment,15 the loss of a methyl group (14 Da) must have occurred in the cyclohexane moiety, suggesting the peak to be 2 (Supporting Information, Figure S5). In these gene inactivation experiments, a new FK506 biosynthetic intermediate 4 and the previously known intermediates 2 and 3 were detected. We then tried to detect the possible intermediates 9-hydroxyFK506 (5) and 9-hydroxy-31-O-demethylFK506 (6) from the extract of wild-type strain. Although compounds 5 and 6 could not be detected in the crude extract, analysis of partially purified FK506-enriched fraction by HPLC−ESI-MS/MS suggested the presence of 4 as a minor compound and a trace amount of 5 (Figure 2D). In the HPLC−ESI-MS/MS analysis, a peak eluted at 18 min with its ammonium adduct ion at m/z 823.3 (Figure 2D), and the MS/MS spectrum of this precursor ion showed fragment ions at 788.2, 770.2, 752.0, and 578.0 (Supporting Information, Figure S26). The appearance of a fragment ion at m/z 578.0 corresponding to the addition of 2 Da from the diagnostic fragment of FK506 at m/z 576.0 suggested that one of the carbonyl groups in the C1−C24 fragment might be replaced with a hydroxyl group.15 As the peak eluted at 18 min was more polar than FK506 (tR 25 min), it was assumed to be 5 by ruling out the possibility of dehydrogenation of a double bond in FK506.15 However, 6 could not be detected either in the crude extract or after its fractionation. Unfortunately, a trace amount of 5 in wild-type strain did not allow us to obtain an amount sufficient to measure the NMR spectroscopic data. Thus, to isolate the proposed biosynthetic intermediates 2 from ΔfkbMapr, 3 from ΔfkbDapr, and 4 from ΔfkbDin‑frame, their fermentation was scaled up to yield sufficient material to elucidate their structures by NMR analysis. After culturing these strains, broths were subjected to suitable postharvesting procedures to prepare crude extracts (see the Supporting

which undergoes hydroxylation and probably further oxidation by the action of FkbD to yield 2, which can finally be methylated by FkbM to give FK506 (Figure 1A). However, the functional assignment of FkbD for the oxidation reaction at the C9-position is incomplete. In our previous study on the profiling of FK506 biosynthetic intermediates using HPLC−ESI-MS/MS, we detected 9deoxoFK506 (4) and 9-hydroxyFK506 (5) as possible intermediates based on MS/MS fragmentation patterns.15 These results suggested the existence of an alternative postPKS modification pathway to the previously proposed pathway (Figure 1A). Hence, the present study was undertaken to characterize the sequence of the C9-oxidation and 31-Omethylation reactions, and the possible intermediates involved in the final post-PKS modification in FK506 biosynthesis. Two parallel pathways may exist using the 9-deoxo-31-OdemethylFK506 (3) as a common starting point. First, FkbD catalyzes the hydroxylation at the C9-position of 3 to form 9hydroxy-31-O-demethylFK506 (6), which undergoes further oxidation to yield 31-O-demethylFK506 (2) by FkbD. Then, FkbM performs 31-O-methylation to afford FK506 (Figure 1A). Second, 3 might be converted to 9-deoxoFK506 (4) by the action of FkbM, which then undergoes hydroxylation to form 9-hydroxyFK506 (5), followed by oxidation of the hydroxyl group by FkbD to finally give FK506 (Figure 1B). This report explores the parallel pathways of post-PKS modification to yield FK506 by gene inactivation, the identification of five FK506 biosynthetic intermediates by detailed HPLC−ESI-MS/MS, HR-MS, NMR analysis, and in vitro enzymatic reactions. The detailed understanding of the post-PKS modification steps for the biosynthesis of FK506 presented here has allowed us to discover the previously undescribed intermediates and remarkable parallel pathways leading to FK506.

■

RESULTS AND DISCUSSION As a starting point, each f kbD or f kbM gene in Streptomyces sp. KCTC11604BP was inactivated by in-frame deletion or gene replacement using a PCR-targeting method. The f kbD gene in the wild-type strain was inactivated by in-frame deletion to avoid any polar effect on the expression of downstream genes. The HPLC−ESI-MS/MS analysis of the organic extract of the resulting ΔfkbDin‑frame strain showed a major peak eluted at 28 min with m/z 807.1 (Figure 2A). The MS/MS spectrum derived from this ammonium adduct ion m/z 807.1 yielded fragment ions at m/z 772.0, 754.0, 736.1, and 561.9 (Supporting Information, Figure S19). The difference of 14 Da with the ammonium adduct ion of FK506 at m/z 821.3 and its diagnostic fragments at m/z 804.1, 786.2, 768.2, 750.1, and 576.0 (Supporting Information, Figure S4) suggest that the new intermediate was deficient in the keto group at the C9position, and would be 9-deoxoFK506 (4) because the fragment ion at m/z 576.0 of FK506 results from the C1− C24 fragment (polyketide backbone along with pipecolate moiety; in the case of 4 it was observed at m/z 561.9).15 This supports the role of FkbD in the oxidation process. The mutant (ΔfkbDapr strain), in which fkbD was inactivated by gene replacement with an apramycin resistance cassette, produced 1094

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Information) and then processed by repeated chromatographic separation to afford sufficiently pure compounds to determine their structures using MS and NMR analysis. Although the structure of biosynthetic intermediate 31-OdemethylFK506 (2) has previously been reported along with its 1 H and 13C NMR data, its NMR peaks were not fully assigned.14,16−18 Therefore, we tried to confirm the structure of compound 2 by recording full 1D and 2D NMR data followed by its in-depth analysis (Table 1; Supporting Information, Figures S6−S11). The molecular formula of 2 was established as C43H67NO12 on the basis of HR-MS data showing potassium adduct ion at m/z 828.4326 [M + K]+ (calcd m/z 828.4300). Similarly the structure of 9-deoxo-31-O-demethylFK506 (3) was previously determined on the basis of MS and either 1H NMR14 or 13C NMR19 spectroscopic analysis without detailed assignments. Its HR-MS spectrum showed a quasi-molecular ion peak [M + K]+ at m/z 814.4510 (calcd m/z 814.4508), corresponding to the molecular formula C43H69NO11. The 1D and 2D NMR spectra (Supporting Information, Figures S13− S18) were acquired for 3, and subsequent detailed study of the NMR data (Table 1) confirmed the structure of 3 as 9-deoxo31-O-demethylFK506. Compound 4 has previously been synthesized and its structure characterized on the basis of partially assigned 1H and 13C NMR data,19−21 but its occurrence as a natural biosynthetic intermediate in FK506producing strains had never been reported. HR-MS analysis yielded a [M + K]+ ion at m/z 828.4677, consistent with a molecular formula of C44H71NO11 (calcd m/z 828.4664). To confirm its structure, detailed 1D and 2D NMR analysis was performed (Table 1) and results were compared with reported data.20 The 1H NMR spectrum (Supporting Information, Figure S20) showed characteristic signals for the FK506 skeleton, comprising three doublets for methyl groups at δH 0.93/H3-38, 0.76/H3-39, and 0.87/H3-41; two methyl singlets at δH 1.65/H3-40 and 1.67/H3-42; three methoxy singlets at δH 3.35/H3-44, 3.36/H3-43, and 3.39/H3-45; and a multiplet of an olefinic proton at δH 5.71/H-36 (Table 1). The 13C NMR spectrum (Supporting Information, Figure S21) contained 44 signals that accounted for all the expected carbons. However, careful analysis of the 13C NMR spectrum of 4 indicated that the signal in the region δC 189−196 (C9 of FK506) had disappeared while an additional signal at δC 37.5 was observed. The HSQC spectrum (Supporting Information, Figure S23) showed that proton signals at δH 2.67 (d, J = 15.5 Hz) and 2.49 (d, J = 15 Hz) were correlated with the carbon signal at δC 37.5. In the HMBC spectrum, correlations (Supporting Information, Figures S24 and S25) were observed from proton signals at δH 2.67 (d, J = 15.5 Hz) and 2.49 (d, J = 15 Hz) to both carbon centers, C8 (δC 174.2) and C10 (δC 98.7), therefore suggesting that the C9-keto group was replaced by a methylene group. The COSY spectrum (Supporting Information, Figure S22) of 4 showed 6 spin systems, which were connected on the basis of HMBC correlations (Supporting Information, Figures S24 and S25). The positions of methyl and methoxy groups were also assigned on the basis of respective HMBC correlations from protons to their neighboring carbon centers. The stereochemistry was assumed to be the same as that of the parent compound FK506. This is the first report of the structure elucidation of compound 4 employing a combination of 1D and 2D NMR and HR-MS spectroscopic data. It is usual to observe complex NMR spectra for FK506 and related macrolides due to the presence of amide bond rotamers (Figure 3) of hemiketal tautomers,22,23 and the isolated FK506 intermediates (2−4)

Figure 3. Structures of the major and minor rotamers for FK506 (1).

exhibited such major and minor signals in the 1H and 13C NMR spectroscopic data. In the case of compound 2, both 1H and 13 C NMR spectra (Supporting Information, Figures S6 and S7) exhibited signals for the major (60%) and minor (40%) rotamers as the presence of the C9-keto group causes different arrangements of these rotamers in space. The absence of the C9-keto group in the compounds 3 and 4 results in little effect on the proton chemical shift values of both rotamers rendering them inseparable, and only the 13C NMR spectra of compounds 3 (Supporting Information, Figure S14) and 4 (Supporting Information, Figure S21) displayed distinguishable signals for the amide bond rotamers (major rotamer 90%, minor rotamer 10%). Hence, the signals for the minor rotamer of 2 were assigned but those for 3 and 4 could not be (Table 1). Because previous studies had reported weaker immunosuppressive activity for 31-O-demethyl, 9-deoxo, and 9-hydroxy derivatives of FK50614,16 compared to FK506 itself, we did not evaluate the biological activity of compounds 2, 3, and 4. The characterization of three out of the five suggested biosynthetic intermediates supported the proposed parallel pathways shown in Figure 1. The in vitro reaction using the recombinant FkbD and FkbM to confirm the suggested parallel post-PKS modification pathways for FK506 biosynthesis was conducted. The recombinant FkbD and FkbM enzymes were expressed in Escherichia coli as histidine-tagged proteins, and purified by nickel-affinity chromatography. On the basis of the in vivo deletion results, 9-deoxo-31-O-demethylFK506 (3) and 9-deoxoFK506 (4) were chosen as substrates for the in vitro assay of FkbD and their reactions were monitored by HPLC− ESI-MS/MS. As shown in Figure 4A, FkbD was shown to utilize 3 as a substrate in the presence of NADPH, and to convert it to the corresponding hydroxylated product (probably compound 6) and the further oxidized 31-O-demethylFK506 (2). After further incubation with FkbD, 3 was almost completely converted into 2. In the FkbD reaction with 3, a compound, expected to be compound 6, was detected with m/z 809.3, 16 Da higher than the molecular weight of 3. The MS/ MS spectrum derived from this ammonium adduct ion m/z 809.3 provided fragment ions at m/z 792.1, 774.3, 756.2, 738.2, and 578.0. As discussed above, the characteristic fragment ion at m/z 578.0 resulting from the C1−C24 fragment of 6 (m/z 576.0 for the C1−C24 fragment of FK506) suggested that the C9-keto group was replaced by a hydroxyl group. The 16 Da increase compared to 3 (m/z 793.1; 6, m/z 809.3) also corroborated the presence of a hydroxyl group at the C9position, supporting that this new compound might be 9hydroxy-31-O-demethylFK506 (6) (Supporting Information, Figure S27). Its HR-MS spectrum was obtained by directly analyzing the reaction mixture of FkbD with the substrate 3 and selecting its quasi-molecular ion. The molecular formula was 1095

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Figure 4. FkbD and FkbM activity assays in vitro. (A) In vitro conversion of 9-deoxo-31-O-demethylFK506 (3) to 9-hydroxy-31-O-demethylFK506 (6) and 31-O-demethylFK506 (2) by FkbD; 3 as substrate with the boiled FkbD (I), the active FkbD for 1 h reaction (II), and the active FkbD for 3 h reaction (III). (B) In vitro conversion of 9-deoxoFK506 (4) to 9-hydroxyFK506 (5) and FK506 (1) by FkbD; 4 as substrate with the boiled FkbD (IV), the active FkbD for 1 h reaction (V), and the active FkbD for 3 h reaction (VI). (C) In vitro conversion of 9-deoxo-31-O-demethylFK506 (3) to 9-deoxoFK506 (4) by FkbM; 3 as substrate with the boiled FkbM (VII), and the active FkbM for 3 h reaction (VIII). (D) In vitro conversion of 31-O-demethylFK506 (2) to FK506 (1) by FkbM; 2 as substrate with the boiled FkbM (IX), and the active FkbM for 3 h reaction (X).

determined to be C43H69NO12 on the basis of molecular ion at m/z 830.4467 [M + K]+ (calcd m/z 830.4457). Thus, the identity of 6 was also supported by HR-MS data. Similarly, FkbD reaction with 9-deoxoFK506 (4) resulted in the appearance of a compound expected to be 9-hydroxyFK506 (5) and FK506, and over the course of time, 4 was consumed and FK506 accumulated (Figure 4B). A compound expected to be 5 was detected with m/z 823.3, which is 16 Da higher than the molecular weight of 4. HR-MS for 5 was also acquired by directly analyzing the reaction mixture containing FkbD and substrate 4. Its molecular formula was determined to be C44H71NO12, affording an [M + K]+ ion at m/z 844.4616 (calcd m/z 844.4613). 9-HydroxyFK506 (5) and 9-hydroxy-31-OdemethylFK506 (6) were detected as new biosynthetic intermediates in in vitro assay, which supported the role of

FkbD in double oxidation steps at the C9-position, hence ruling out the need for the involvement of other oxygenase enzymes in the C9-oxidation step. The SAM-dependent FkbM reactions were also assessed using 31-O-demethylFK506 (2) and 9deoxo-31-O-demethylFK506 (3) as substrates, which were converted to FK506 and 9-deoxoFK506 (4), respectively, as proposed (Figures 4C and 4D). In addition, it seems that FkbD has a similar substrate preference for 3 and 4 based on the conversion yields. Similarly, there appears to be no significant difference in the substrate preference of FkbM between 2 and 3. Therefore, it is plausible that the two independent biosynthetic pathways shown in Figure 1 proceed in parallel at similar rates, although detailed kinetic analysis is further required to identify the major post-PKS modification pathway to FK506. 1096

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m/z 790.1, 772.1, 754.0, 736.1, 576.0; (+)-HR-MS m/z 828.4326 [M + K]+ (calcd for C43H67KNO12, 828.4300). 9-Deoxo-31-O-demethylFK506 (3): amorphous white powder; 1 H and 13C NMR (see Table 1); (+)-ESIMS m/z 793.1 [M + NH4]+; (+)-MS/MS m/z 776.1, 758.0, 740.0, 722.0, 561.9; (+)-HR-MS m/z 814.4510 [M + K]+ (calcd for C43H69KNO11, 814.4508). 9-DeoxoFK506 (4): amorphous white powder; 1H and 13C NMR (see Table 1); (+)-ESIMS m/z 807.1 [M + NH4]+; (+)-MS/MS m/z 772.0, 754.0, 736.1, 561.9; (+)-HR-MS m/z 828.4677 [M + K]+ (calcd for C44H71KNO11, 828.4664). 9-HydroxyFK506 (5): (+)-ESIMS m/z 823.3 [M + NH4]+; (+)-MS/MS m/z 788.2, 770.2, 752.0, 578.0; (+)-HR-MS m/z 844.4616 [M + K]+ (calcd for C44H71KNO12, 844.4613). 9-Hydroxy-31-O-demethylFK506 (6): (+)-ESIMS m/z 809.3 [M + NH4]+; (+)-MS/MS m/z 792.1, 774.3, 756.2, 738.2, 578.0; (+)-HRMS m/z 830.4467 [M + K]+ (calcd for C43H69KNO12, 830.4457). In Vitro Characterization of FkbD and FkbM. The details regarding the preparation and purification of recombinant FkbD and FkbM are described in the Supporting Information. Recombinant FkbD (1 mM) was incubated with 9-deoxo-31-O-demethylFK506 (3) or 9-deoxoFK506 (4) in reaction buffer (50 mM sodium phosphate, pH 7.4, 1 mM NADPH, 50 μg/mL ferredoxin, 0.1 U of ferredoxin reductase) at 30 °C for 1 h or 3 h. FkbM (1 mM) was incubated with 31-O-demethylFK506 (2) or 9-deoxo-31-O-demethylFK506 (3) in reaction buffer (50 mM potassium phosphate buffer, pH 7.4, 5 mM SAM, 5 mM MgSO4) at 30 °C for 3 h. Reactions were monitored using HPLC−ESI-MS/MS analysis, as previously described.15

In summary, we have discovered the parallel pathways responsible for the postmodification step in the biosynthesis of FK506. Our results clearly demonstrate that there are two independent biosynthetic routes to FK506, and it has been shown that 9-deoxoFK506 derivatives, 9-deoxo-31-O-demethylFK506 (3) and 9-deoxoFK506 (4), can be used as substrates for FkbD, whereas FkbM can utilize 31-O-demethyl derivatives, 31-O-demethylFK506 (2) and 9-deoxo-31-OdemethylFK506 (3), as substrates. These substrate-flexible post-PKS modification enzymes, FkbD and FkbM, can provide a potential tool for the combinatorial biosynthesis of novel macrolide derivatives.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were acquired using a Varian INOVA 500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C nuclei. Chemical shifts are given in ppm using tetramethylsilane (TMS) as an internal reference. All NMR data processing was done using Mnova software (Mestrelab Research S.L.). Samples for NMR analysis were prepared by dissolving each compound in 250 μL of CDCl3 (Sigma) and placing the solutions in 5 mm Shigemi advanced NMR microtubes (Sigma) matched to the solvent. HPLC−ESI-MS/MS spectra using an ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm; Waters) column were recorded on a Waters/Micromass Quattro micro MS interface consisting of a Waters 2695 separation module connected directly to a Micromass Quattro micro MS. The HR-MS data was obtained using a Waters SYNAPT G2-S mass spectrometer coupled with UPLC. HPLC purification was performed using preparative Spherisorb S5 ODS2 (Waters, 250 × 20 mm, 5 μm) and semipreparative Watchers 120 ODS-BP (250 × 10 mm, 5 μm) columns on an Acme 9000 HPLC system (YL Instrument Co. Ltd., Korea) consisting of a SP930D gradient pump coupled with a UV730D UV detector set to 205 nm and a CTS30 column oven set to 50 °C. Construction of Mutants and Culture Conditions. The f kbD and f kbM genes were inactivated in the FK506-producing strain Streptomyces sp. KCTC11604BP by in-frame deletion or gene replacement with an apramycin-resistance gene via double crossover homologous recombination. Details regarding DNA manipulation and construction of plasmids for gene deletion and heterologous expression as well as the resulting mutant strains are described in the Supporting Information (see also Table S1). Spores of Streptomyces sp. KCTC 11604BP and its gene deletion mutants were generated on ISP4 agar plates, and a seed culture was prepared in R2YE24 broth. 50 mg of vegetative cells grown in the seed culture were inoculated into 250 mL baffled flasks containing 50 mL of R2YE medium and cultivated on an orbital shaker (set at 180 rpm) for 6 days at 28 °C. Escherichia coli BL21(DE3) and E. coli BL21(DE3)pLysS (Novagen) were used as heterologous hosts for the expression of recombinant FkbD and FkbM. The E. coli strains were grown in LB liquid medium. Ampicillin (100 μg/mL), apramycin (50 μg/mL), and kanamycin (50 μg/mL) were selectively added to the growth media as required. Extraction and Isolation. Wild-type strain Streptomyces sp. KCTC11604BP and mutant strains were cultured in production media and processed separately. The individual culture broths of each strain were centrifuged, and the supernatant was subjected to solvent− solvent partition with ethyl acetate. The ethyl acetate extracts were evaporated, and the resultant brown residues were either separated by open column chromatography or directly injected to preparative reversed-phase HPLC to obtain fractions containing target compounds. These partially separated fractions were subjected to semipreparative reversed-phase HPLC to afford individual compounds, which were again purified using semipreparative reversedphase HPLC to yield pure compounds. The details regarding the isolation and purification of products obtained are described in the Supporting Information. 31-O-DemethylFK506 (2): amorphous white powder; 1H and 13C NMR (see Table 1); (+)-ESIMS m/z 807.1 [M + NH4]+; (+)-MS/MS

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details; tables for bacterial strains, plasmids, and primers used in this study; SDS−PAGE analysis of purified proteins; and ESI-MS/MS, 1D NMR, and 2D NMR spectra of 1−6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-3277-4446. Fax: +82-2-3277-3419. Author Contributions ‡

Y.H.B. and P.B.S. contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Research Foundation (NRF), funded by the Korean government (MEST) (20120006243 and 20120000650); the Intelligent Synthetic Biology Center of the Global Frontier Project, funded by the MEST (2012054879); and the Seoul R&BD Program (ST110024).

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REFERENCES

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