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Biochemical Characterization of a Malonyl-Specific Acyltransferase Domain of FK506 Biosynthetic Polyketide Synthase Yue-Yue Wanga†, Long-Fei Baia†, Xin-Xin Rana, Xin-Hang Jianga, Hui Wuc, Wei Zhangc, Mei-Ying Jinc, Yong-Quan Lia,b and Hui Jianga,b* a
College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China; bKey Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province, Hangzhou, Zhejiang 310058, China; cHangzhou Zhongmei Huadong Pharmaceutical Co. Ltd, Hangzhou, Zhejiang 310011, China Abstract: Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions.
Keywords: Acyltransferase, FK506, malonyl-CoA, methylmalonyl-CoA, polyketide synthase. INTRODUCTION Polyketide natural products, some of which are commonly used as medicines or biological active agents, are biosynthesized from acyl units by polyketide synthases (PKSs) [1-4]. PKSs can be classified into three types based on their enzyme structures. The type I PKSs are organized into modules, each of which harbors a set of domains. Each elongation module of typical type I PKSs minimally contains a ketoacyl synthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain [5-8]. ATless type I PKSs are atypical type I PKSs, which lack AT domains in elongation modules and contain at least one discrete AT providing the missing AT activities in trans [9-11]. Iterative type I PKSs are other atypical type I PKSs, whose domains perform activities iteratively [12-14]. Each type II PKS minimally contains a KS, a KS (also named as chainlength factor), and an ACP [15-17]. Each type III PKS contains only one KS, which acts on the acyl-CoAs directly, independent of ACP [18, 19]. ATs are essential for the polyketide biosynthesis catalyzed by type I PKSs. To date, the functions of ATs are known to transfer acyl units from acyl-coenzyme A (CoA) or acyl-S-ACP into holo-ACPs, initiating the incorporation of acyl units into polyketides [20-21]. ATs transfer acyl units into ACPs via self-acylation reactions and transacylation reactions: (i) ATs transfer acyl units from acyl-CoAs or acylS-ACPs to active Ser residues of ATs to form acyl-O-AT intermediates and CoA; (ii) ATs then transfer acyl units from *Address correspondence to this author at the College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China; Tel: (+86)571-88208569; E-mail:
[email protected] † These authors contributed equally to this work. 0929-8665/15 $58.00+.00
acyl-O-ATs to holo-ACPs to form acyl-S-ACPs [10, 22]. The acyl specificity of an AT determines the molecular structure of final product. FK506, an important immunosuppressive medicine, is biosynthesized by a hybrid of type I PKS and non-ribosomal peptide synthetase (NRPS) [23-26]. In this in vitro study, we characterized the specificity of AT10FkbA of the FK506 biosynthetic PKS from Streptomyces tsukubaensis YN06, whose function is proposed to transfer malonyl units from malonyl-CoA into ACP10FkbA. Our results showed: (i) AT10FkbA transferred both malonyl and methylmalonyl units to itself in the self-acylation reaction, forming malonyl-OAT10FkbA and methylmalonyl-O-AT10FkbA, respectively; (ii) competition of malonyl-CoA and methylmalonyl-CoA showed that the malonyl unit was the favored substrate of AT10FkbA in the self-acylation reaction; (iii) AT10FkbA transferred only the malonyl unit but not any methylmalonyl unit into ACPs in the transacylation reaction (Fig. 1). AT10FkbA represents the ATs which control the acyl specificity in the transacylation reactions. MATERIAL AND METHODS 2.1. Production of AT10FkbA, ACP10FkbA, and ACP2FkbB in E. coli. The AT10FkbA, ACP10FkbA, and ACP2 FkbB genes were amplified by PCR from the genomic DNA of S. tsukubaensis YN06 using the following primers: for AT10FkbA (forward) G CGC CAT ATG CCC GCC GCT CCC GTA CCG CT/(reverse) GC GCA AGC TTA CCC GGA GCC GCC GGC CGG TT, for ACP10FkbA (forward) G CGC CAT ATG GAC GGC GGA CGC CCG GTG GAC/(reverse) GCG CAA GCT TAG GTG GGC GGG TCG TCG TCC GC, and © 2015 Bentham Science Publishers
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Figure 1. Proposed pathway of loading of acyl unit into ACP10FkbA catalyzed by AT10FkbA via a self-acylation reaction (A) and a transacylation reaction (B). M, malonyl; MM, methylmalonyl; AT, AT10FkbA; ACP, ACP10FkbA.
for ACP2FkbB (forward) G CGC CAT ATG ACG CCC GCA CCG GCC GGC TC/(reverse) GCG CAA GCT TCA ACG GTC GGG GTC GGG GTC GT. The PCR products were cloned as NdeI/HindIII fragments into pET28a and sequenced to confirm PCR fidelity, resulting in overexpression plasmids pHJ0051-pHJ0053, respectively. E. coli BL21(DE3) containing each of the overexpression plasmids was grown at 30 ºC until the OD600 reached 0.5. IPTG was then added at the final concentration of 0.1 mM, and incubation continued at 30 ºC for 6 h. The produced N-His6-tagged proteins were purified by Ni-NTA agarose (QIAgen, Valencia, CA). 2.2. Formation of Acyl-O-AT10FkbA A typical reaction mixture of 50 μL, containing 20 μM AT10FkbA, 200 μM malonyl-CoA (or methylmalonyl-CoA), 10 mM Tris-HCl (pH 8.0), was incubated at 25 ºC for 1 h. In the assay of competition of acyl-CoAs, both malonyl-CoA and methylmalonyl-CoA were added together to the final concentration of 200 μM. The reaction mixture was digested with 200 ng trypsin in the presence of 10 mM NH4HCO3 at 37 ºC for 3 h. 2.3. Biochemical Synthesis of holo-ACPs A typical reaction mixture of 50 μL, containing 16 μM apo-ACP, 160 μM CoA, 2 μM Sfp, 1.25 mM MgCl2, 100 mM Tris-HCl (pH 8.0), was incubated at 25 ºC for 30 min. 2.4. Production of Acyl-S-ACPs To the above reaction mixture, AT10FkbA and malonylCoA (or methylmalonyl-CoA) were added to the final concentration of 1.6 μM and 160 μM, respectively. The reaction mixture was incubated at 25 ºC for 1 h. 2.5. LC-MS Analyses of Proteins HPLC separation was carried out on an Agilent SB-C18 column (3.5 μm, 80 Å, 2.1 150 mm) at 35°C. Solvent A was water with 0.1% formic acid. Solvent B was acetonitrile. The following binary gradient was used: a linear gradient from 10% to 30% solvent B from 0 to 5 min, a linear gradient from 30% to 50% solvent B from 5 to 55 min, a linear gradient from 50% to 70% solvent B from 55 to 60 min, and equilibration to initial conditions for 13 min at a flow rate of
0.2 ml/min. UV detection was performed at both 220 nm and 280 nm. MS with an electrospray ionization (ESI) source was performed as follows: positive mode, source voltage of 2.5 kV, capillary voltage of 41 V, sheath gas flow of 45 arbitrary units, auxiliary/sweep gas flow of 5 arb, and capillary temperature of 330 °C. RESULTS 3.1. Production of AT10FkbA, ACP10FkbA, and ACP2FkbB in E. coli. The whole genomic DNA of S. tsukubaensis YN06, an industrial FK506 producer isolated from Yunnan China, was sequenced to identify the FK506 biosynthetic gene cluster. The FK506 biosynthetic gene cluster in S. tsukubaensis YN06 shows identical domain organization and high homology with the gene cluster in Streptomyces sp. KCTC11604BP [23]. The proposed biosynthetic pathway of FK506 involves incorporation of malonyl units, methylmalonyl units, allylmalonyl units, and methoxymalonyl units, which are transferred to the PKS machinery catalyzed by ten ATs. According to the relationship of the molecular structure of FK506 and the domain organization of FK506 biosynthetic PKS, AT10FkbA is predicted to transfer malonyl units into ACP10FkbA from malonyl-CoA [23-26]. AT10FkbA contains a conserved motif HAFH found in malonyl specific ATs [20, 27-28], which is consistent with the above prediction (Fig. 2B). To characterize the specificity of ATs, AT10FkbA was produced as a N-His6-tagged protein in E. coli BL21(DE3) and purified by affinity chromatography on Ni2+-NTA resin (Fig. 2A). To obtain the ACP substrates of AT10FkbA, both ACP10FkbA and ACP2FkbB were also produced in E. coli and purified (Fig. 2A). LC-MS data showed that these two ACPs were in apo-forms. The holo-ACPs were obtained by phosphopantetheinylation of the apo-ACPs catalyzed by the known promiscuous phosphopantetheinyl transferase Sfp [29] and confirmed by LC-MS. 3.2. Self-acylation of AT10FkbA AT10FkbA contains a conserved motif GHSVG found in the ATs: GHSxG, in which the active Ser residue covalently links the acyl unit to form an acyl-O-AT intermediate [20]. To detect the formation of acyl-O-AT, AT10FkbA was sub-
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Figure 2. (A) Purified proteins on 15% SDS-PAGE. Lane M, protein markers; lane 1, AT10FkbA; lane 2, ACP10FkbA; lane 3, ACP2FkbB. (B) The protein sequence of AT10FkbA. The active-site containing peptide fragment generated by trypsin digestion is printed in blue. The active Ser residue is printed in red. The malonyl specific motif HAFH is printed in green.
jected to trypsin digestion and LC-MS analysis. LC-MS data identified the active Ser containing peptide fragment LLDHWGVRPDVVVGHSVGEVTAAHAAGVLTLTDA TR (the calculated molecular weight: 3,721, the found molecular weight: 3,720) (Fig. 2B and 3A). AT10FkbA was then incubated with its native substrate malonyl-CoA. Then the reaction mixture was digested with trypsin and then analyzed by LC-MS. LC-MS data showed that the molecular weight of a fraction of the above peptide fragment was increased by 86 Da, indicating the formation of the native intermediate malonyl-O-AT10FkbA. Recognizing that the peak height ratio in mass spectrometry is not precisely quantitative to the relative amounts of the analytes, the malonylation conversion yield was estimated to be 44% by comparing ion peak heights of the acylated peptide and the intact peptide in the mass spectrum (Fig. 3B). Surprisingly, the replacement of malonyl-CoA with nonnative methylmalonyl-CoA in the above reaction resulted in increased molecular weight of a fraction of the above peptide fragment by 100 Da, indicating the formation of the nonnative intermediate methylmalonyl-O-AT10FkbA. Under reaction conditions identical to those applied for malonylation, the methylmalonylation yield was similarly estimated to approximately 44% (Fig. 3C). The above results indicate that AT10FkbA can transfer both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction. 3.3. Competition of Acyl-CoAs in the Self-Acylation Reaction To further characterize which acyl-CoA was the preferred substrate of AT10FkbA in the self-acylation reaction, AT10FkbA was incubated with malonyl-CoA and methylmalonyl-CoA with the ratio of 1:1 and then digested with trypsin. LC-MS analysis showed that AT10FkbA was both malonylated and methylmalonylated. In the competition reaction of malonyl-CoA and methylmalonyl-CoA, the malonylation yield was about twice of the methylmalonylation yield (about 17% vs. about 8%, respectively) (Fig. 3D).
These results support that the native substrate malonyl-CoA is the preferred substrate of AT10FkbA in the self-acylation reaction. 3.4. Transacylation of holo-ACP10FkbA Catalyzed by AT10FkbA Since AT10FkbA has broad specificity for acyl-CoAs in the self-acylation reaction, we characterized whether it had strict specificity for acyl-CoAs in the transacylation reaction. The above biochemically synthesized holo-ACP10FkbA was incubated with malonyl-CoA in the presence of AT10FkbA. LC-MS analysis showed that a fraction of holo-ACP10FkbA was converted to malonyl-S-ACP10FkbA. The production of malonyl-S-ACP10FkbA was abolished in the absence of AT10FkbA, confirming that the malonylation of holoACP10FkbA was AT10FkbA dependent (Fig. 4A and B). These results are consistent with the proposed function of AT10FkbA that is transferring malonyl units into ACP10FkbA. Replacement of malonyl-CoA with methylmalonyl-CoA in the above reaction failed to result in the production of methylmalonyl-S-ACP10FkbA, confirming that AT10FkbA has strict specificity for malonyl-CoA but not methylmalonylCoA in the transacylation reaction (Fig. 4E and F). 3.5. Transacylation of Holo-ACP2FkbB Catalyzed by AT10FkbA According to the relationship of the molecular structure of FK506 and the domain organization of FK506 biosynthetic PKS, ACP2FkbB is predicted to be loaded with methylmalonyl units catalyzed by AT2FkbB. To characterize whether AT10FkbA has broad specificity for ACPs, the holo-ACP2FkbB was incubated with malonyl-CoA in the presence of AT10FkbA. After incubation of holo-ACP2FkbB, LC-MS data showed that a fraction of holo-ACP2FkbB was converted to malonyl-S-ACP2FkbB. The production of malonyl-SACP2FkbB was abolished in the absence of AT10FkbA, confirming that the malonylation of holo-ACP2FkbB was
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Figure 3. LC-MS analyses of trypsin digested AT10FkbA. (A) AT10FkbA; (B) Incubation of AT10FkbA with malonyl-CoA; (C) Incubation of AT10FkbA with methylmalonyl-CoA; (D) Incubation of AT10FkbA with malonyl-CoA: methylmalonyl-CoA (1:1). M, malonyl; MM, methylmalonyl; AT, AT10FkbA; AT10Frag, the active Ser containing peptide fragment of AT10FkbA.
AT10FkbA dependent (Fig. 4C and D). Replacement of malonyl-CoA with methylmalonyl-CoA in the above reaction failed to result in the production of methylmalonyl-SACP2FkbB (Fig. 4G and H). These results support that the specificity for the acyl unit is only controlled by AT but not ACP in the transacylation reaction. DISCUSSION Only a few ATs transfer different acyl units into ACPs, indicating they have broad specificity for acyl units in both self-acylation reactions and transacylation reactions. PikAIV-AT6 in pikromycin PKS loads both native methylmalonyl units and non-native ethylmalonyl units but neither non-native malonyl units nor non-native propionyl units into ACP [30]. EpoAT3 in epothilone PKS loads both malonyl and methylmalonyl units into ACP, resulting in the production of epothilone analogues [28]. Most ATs transfer only native acyl units into ACPs, suggesting they have strict specificity for acyl units. Among them, some ATs have been characterized to have strict specificity in self-acylation reactions, indicating these ATs control the acyl specificity in the first half-reactions. All of the six ATs in 6-deoxyerythronolide B synthase (DEBS) load only the native (2S)-methylmalonyl unit but not the non-native (2R)-methylmalonyl unit [22]. Kinetic analysis of the AT3 in DEBS shows that the acyl-O-AT formation rate is about 200fold higher for the native substrate methylmalonyl-CoA than for the non-native substrate malonyl-CoA [31]. However, it
is uncertain whether all ATs control their acyl specificity in the self-acylation or the transacylation reactions. Recently, some ATs with strict specificity for acyl units have been characterized to have broad specificity in selfacylation reactions, indicating that these ATs control the acyl specificity in the second half-reactions. Although ZmaF transfers only aminomalonyl unit into ZmaA-ACP1, ZmaF can form aminomalonyl-O-ZmaF, seryl-O-ZmaF, and 2amino-3-oxo-propionyl-O-ZmaF intermediates [32]. Although ZmaA-AT transfers only hydroxymalonyl units into ZmaA-ACP2, ZmaA-AT can form aminomalonyl-O-ZmaAAT intermediates [32]. Structural and biochemical study of ATDYN10 of DynE8 shows that ATDYN10 transfers only malonyl units into ACP but ATDYN10 also hydrolyzes acetyl-CoA via an acetyl-O-ATDYN10 intermediate [27]. Our results here support that AT10FkbA has broad specificity in the first halfreaction and controls acyl specificity in the second halfreaction. The formation of methylmalonyl-O-AT10FkbA decreases the effective amount of AT10FkbA and is inhibited by the presence of malonyl-CoA. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS H. Jiang and Y.-Q. Li designed research. Y.-Y. Wang, L.-F. Bai, and, H. Jiang performed research. X.-X. Ran and
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Figure 4. LC-MS analyses of acylation of ACPs. Incubation of ACP10FkbA with malonyl-CoA in the presence of AT10FkbA (A) and in the absence of AT10FkbA (B). Incubation of ACP2FkbB with malonyl-CoA in the presence of AT10FkbA (C) and in the absence of AT10FkbA (D). Incubation of ACP10FkbA with methylmalonyl-CoA in the presence of AT10FkbA (E) and in the absence of AT10FkbA (F). Incubation of ACP2FkbB with methylmalonyl-CoA in the presence of AT10FkbA (G) and in the absence of AT10FkbA (H). M, malonyl; MM, methylmalonyl; AT10, AT10FkbA; ACP10, ACP10FkbA; ACP2, ACP2FkbB.
X.-H. Jiang collected data. H. Wu, W. Zhang, and M.-Y. Jin analyzed data. H. Jiang and Y.-Y. Wang wrote paper. This work was financially supported by National Natural Science Foundation of China 31470212 and 31200600 and National High Technology Research & Development Program of China (863 Program) 2012AA02A706 and 2012AA022107.
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