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Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2018 www.elsevier.com/locate/jbiosc
Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4 Yusuke Yamauchi, Yosi Nindita, Keisuke Hara, Asako Umeshiro, Yu Yabuuchi, Toshihiro Suzuki, Haruyasu Kinashi, and Kenji Arakawa* Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan Received 27 January 2018; accepted 9 March 2018 Available online xxx
Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and isolankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A ([sedecamycin) and isolankacidin C. Addition of various divalent metal cations, especially Ca2D, enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biosynthesis; Quinoprotein dehydrogenase; Secondary metabolite; Antibiotics; Pyrroloquinoline quinone]
A group of unique 17-membered carbocyclic polyketide antibiotics lankacidins (Fig. 1) are produced by Streptomyces rochei 7434AN4 that carries three linear plasmids, pSLA2-L, -M, and -S (1). Lankacidins show various biological activities including antimicrobial action against gram-positive bacteria (2). X-ray crystallographic analysis revealed that lankacidin C (1; Fig. 1) and lankamycin (14-membered macrolide antibiotic) inhibit peptide formation synergistically by binding to the neighboring sites in the large bacterial ribosomal subunit (3,4). In addition, lankacidin C enhances stable micro-tubulin assembly and displaces taxoids from their binding sites (5). Nucleotide sequencing and gene inactivation experiments of the largest linear plasmid pSLA2-L revealed that the lankacidin biosynthetic (lkc) gene cluster is located on pSLA2-L (210,614 bp) (6). The lkc cluster contains a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid gene lkcA, three multidomain type-I PKS genes (lkcC, lkcF, lkcG), a discrete dehydratase gene lkcB, a discrete acyltransferase gene lkcD, and an amine oxidase gene lkcE (7). Remarkably, the lkc cluster carries only five ketosynthase (KS) domains although eight condensation reactions of malonyl CoA are necessary, suggesting a modulariterative mixed polyketide biosynthesis of lankacidin (8,9). Heterologous expression of the lkcA-lkcO genes in Streptomyces lividans led to the production of lankacidinol A (2), suggesting that a set of lkc genes is sufficient to construct the lankacidin skeleton (8). Recently, Dickschat et al. (10) reported that an additional dehydratase activity coded on lkcC is required for lankacidin
* Corresponding author. Tel./fax: þ81 82 424 7767. E-mail address: [email protected] (K. Arakawa).
biosynthesis and He et al. (11) speculated the origin of C3 unit (C23C25) amide-bound to the lankacidin skeleton. One of the striking features of the lkc gene cluster is the presence of biosynthetic genes (lkcK-lkcO) for pyrroloquinoline quinone (PQQ; Fig. S1) at its left end (7). This was the first report of the PQQ cluster in gram-positive bacteria. PQQ, the most characterized quinone cofactor, is known as a third family of redox cofactor in various quinoprotein and quinohemoprotein dehydrogenases (12e15). These quino(hemo)proteins utilize quinone cofactor to catalyze dehydrogenation of primary or secondary hydroxyl groups in sugars and alcohols in the periplasm of gram-negative bacteria. Hence we focused on the PQQ biosynthetic cluster lkcK-O (orf8-4) and the quinoprotein dehydrogenase (orf23) gene coded on pSLA2L in S. rochei. Strain FS7, a disruptant of the lkcL (pqqC) gene, showed no inhibitory zone around Rf ¼ 0.5 (CHCl3-MeOH ¼ 15:1, v/v) on TLC bioautography when compared with the parent strain. Its deficiency was restored by exogenous addition of PQQ (7), indicating its crucial role in lankacidin biosynthesis. Nevertheless, a mutant of the orf23 gene showed a distinct inhibitory zone around Rf ¼ 0.5. Here we reinvestigated the metabolic profile of the orf23 disruptant, and describe an extensive biochemical characterization of the quinoprotein dehydrogenase protein involved in lankacidin biosynthesis in S. rochei 7434AN4.
MATERIALS AND METHODS Strains, reagents, and culture conditions All the strains and plasmids used in this study are listed in Table 1. S. rochei strain 51252 that carries only pSLA2-L was used as a parent strain (1). Strain KK23, the orf23 disruptant of strain 51252, was constructed previously (7). YM medium (0.4% yeast extract, 1.0% malt extract, and
1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.03.006
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 1. Structures of lankacidin antibiotics and dehydrogenation step in lankacidin biosynthesis. (A) Structures of lankacidin C (1), lankacidinol A (2), iso-lankacidinol (3), lankacidinol (4), lankacidin A (5), and iso-lankacidin C (6). Ac, acetyl; Me, methyl. (B) Dehydrogenation step catalyzed by Orf23 in lankacidin biosynthesis.
0.4% D-glucose, pH 7.3) was used for antibiotic production. For protoplast preparation and protein expression, S. lividans TK64 and its recombinants were cultured in YEME liquid medium (16). Protoplasts were regenerated on R1M solid medium (17). PQQ disodium salt was purchased from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan).
monitored at 230 nm with a JASCO MD-2010 multi-wavelength photodiode array detector. TLC was developed with CHCl3/MeOH (15:1, v/v) and baked after staining with anisaldehyde-H2SO4. High resolution ESI-MS spectra were measured by a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
Isolation of metabolites Strains were cultivated in YM liquid medium at 28 C for 2 days. The culture broth was extracted twice with equal volume of EtOAc. The combined organic phase was dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was passed through Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) with methanol. The fractions containing lankacidins were combined, and further purified by silica gel chromatography with two different solvent systems of CHCl3-methanol ¼ 50:1e10:1 (v/v) and toluene-EtOAc ¼ 1:3 (v/v). Spectral data including 1H and 13C NMR assignments for compounds 1e4 have already been reported (7,18) (Table S1).
Preparation of the recombinant Orf23 protein A 1.6-kb PCR fragment containing orf23 (nt 41,354-42,985 of pSLA2-L) was amplified using the template cosmid B10 (7) and two primers, 23fNde and 23rHind (Table 1). The PCR fragment was digested with NdeI and HindIII and cloned into pKAR3063H (18), a constitutive expression vector carrying N-terminal (His)6-tag sequence in pHSA81, to give pYY03. The S. lividans TK64 recombinant harboring pYY03 was grown at 28 C for 72 h in YEME liquid medium (34% sucrose) containing 10 mg/ml of thiostrepton. Streptomyces cultures were harvested by centrifugation, and the residual cells were resuspended in 50 mM phosphate buffer (pH 8.0) and disrupted by sonication for three cycles of 20-sec with 0.5-min intervals on ice. The cell-free supernatant was dialyzed when necessary.
Analysis of metabolites Metabolites were analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and electrospray ionization-mass spectrometry (ESI-MS). The crude extract was dissolved in acetonitrile, applied on a reverse-phase HPLC column (Cosmosil Cholester 4.6 x 250 mm; Nacalai Tesque, Kyoto, Japan), eluted with acetonitrile/phosphate buffer (10 mM, pH 8.2) (3:7, v/v) at a flow rate of 0.7e1.0 ml/min, and then
In vitro conversion of lankacidinol in the Orf23 recombinant A standard assay mixture (1 ml) contains 250 mM lankacidinol (4; Fig. 1), 13 mM PQQ, 0.6 mM CaCl2, and the cell-free supernatant of S. lividans TK64/pYY03 (200 ml; prepared from 20-ml culture broth) in 50 mM HEPES buffer (pH 7.5). After incubation at
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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TABLE 1. Bacterial strains, plasmids, and oligonucleotides used in this study. Strains/plasmids/oligonucleotides
Properties/product
Strain S. rochei 7434AN4 51252 KK23 S. lividans TK64 TK64/pYY03 TK64/pHSA81 Plasmid SuperCos-1 cosmid B10 pHSA81 pKAR3063H pYY03 Designed oligonucleotide 23fNde 23rHind
Source/ref.
Wild type (pSLA2-L,M,S) pSLA2-L orf23::kan of pSLA2-L in strain 51252
1 1 7
pro-2, str-6 Strain TK64 with plasmid pYY03, tsr, (His)6-tagged orf23 Strain TK64 with plasmid pHSA81, tsr
16 This study This study
Cosmid vector, bla 45.4-kb pSLA2-L DNA (nt 3,341-48,756) cloned into SuperCos-1 at BamHI site Constitutive expression vector in Streptomyces, tsr Constitutive expression vector in Streptomyces, tsr, N-terminal (His)6-tag sequence 1.6 kb NdeI-HindIII PCR fragment carrying orf23 cloned into pKAR3063H
Stratagene 7 M. Kobayashi 18 This study
50 -CGACATATGAGCACCAGGACCACGCCTG-30 50 -ATTAAGCTTTCAGCGGCCGCGTCTGCAC-30
This study This study
37 C for 2 h, the mixture was extracted with equal volume of EtOAc. The organic phase was concentrated in vacuo and then analyzed by HPLC and ESI-MS. The cell-free supernatant of S. lividans TK64/pHSA81 was also used for negative control. To evaluate the importance of a quinone cofactor, the assay mixture was incubated in the absence of PQQ. Substrate specificity of Orf23 Substrate specificity was estimated by varying the substrate in the standard assay mixture above mentioned. Lankacidinol A (2) and iso-lankacidinol (3; Fig. 1) were used instead of 4 at a concentration of 250 mM. To avoid alkaline hydrolysis of 2, a series of enzymatic assay was carried out at pH 7.0. Metal requirement Metal ion requirement for Orf23 was analyzed by varying the divalent metal ion in the standard assay mixture. Instead of CaCl2, eight metal cations including MgCl2, MnCl2, FeSO4, NiCl2, CoCl2, Ba(OH)2, CuCl2, ZnCl2 were tested at a concentration of 0.6 mM. EDTA (1.0 mM) was also used for a negative control of divalent metal ion.
RESULTS Primary structure of Orf23 The Orf23 protein shows considerable similarities with HR51_10690 protein from Burkholderia cepacia RB-39 (56% identity, 70% similarity) and Sce5403 protein from Sorangium cellulosum So ce56 (43% identity, 56% similarity), both of which were classified as the polyvinyl alcohol dehydrogenase (PVA-DH) family. Amino acid sequences among these proteins together with the most-studied PVA-DH, PvaA, from Pseudomonas sp. VM15C (19) were aligned and compared (Fig. 2). All the proteins contain the anti-parallel b-sheet structures, called W-motifs, that were found in quino(hemo) proteins (13e15). Its consensus sequence, AxDxxxGK(E)xxW, is well conserved in W1, W2, W3, W4, W6, and W7 (Fig. 2). When compared with PvaA, however, Orf23 has neither two adjacent cysteine residues (located between W2 and W3) nor a heme cbinding motif (CxxCH) at the N-terminus. The former makes an eight-membered disulfide ring that causes a hydrophobic interaction with quinone cofactor (20). The latter motif is involved in the binding to heme c, which serves as an electron acceptor from the reduced PQQ prosthetic group (21). Lack of these signature motifs in Orf23 suggests a different catalytic action during dehydrogenation process. Metabolite profile in the orf23 disruptant In our previous study, the orf23 mutant (strain KK23) exhibited a distinct inhibitory zone around Rf ¼ 0.5 (CHCl3-MeOH ¼ 15:1, v/v) on TLC bioautography (7). We now reinvestigated the metabolite profile of strain KK23 by reverse phase HPLC (Fig. 3A). Four peaks with a characteristic UV absorbance at 230 nm (flow rate; 0.7 ml/min) were detected in the parent strain 51252 (Fig. 3A, I); lankacidinol (4) at 6.0 min, iso-lankacidinol (3) at 7.6 min, lankacidin C (1) at
12.1 min, and lankacidinol A (2) at 21.3 min. Whereas strain KK23 showed three peaks of compounds 2e4, but not that of 1 (Fig. 3A, II). The distinct structural difference between lankacidin C (1) and compounds 2e4 is the oxidation degree at C-24 position; ketone in lankacidin C (1) and hydroxyl in compounds 2e4. This finding notified us that Orf23 functions to convert the C23-C25 lactate moiety to pyruvate in lankacidin biosynthesis. Hence, the inhibitory zone detected in strain KK23 in our previous study was not caused by lankacidin C but was by lankacidinol A, whose Rf value was around 0.45 (Fig. 3B). Average yields of compounds 2e4 in strain KK23 were 1.5, 0.4, and 2.5 mg per liter, respectively, while those of compounds 1e4 in strain 51252 were 5.5, 0.6, 0.2, and 0.9 mg per liter, respectively. In vitro bioconversion by Orf23 To study the function of the Orf23 protein in lankacidin biosynthesis, we cloned orf23 into various E. coli expression vectors. However, no protein expression was observed although various culture conditions and vectors were tested. Hence, the orf23 gene was cloned into pKAR3063H (18), a derivative of streptomycete constitutive expression vector pHSA81 (Profs. M. Kobayashi and Y. Hashimoto, personal communication), to afford pYY03. In order to examine the dehydrogenase activity of Orf23, lankacidinol (4) was treated with the cell-free supernatant of S. lividans TK64/pYY03 in the presence of PQQ, and the assay mixture (sample 1) was analyzed by HPLC and ESI-MS. As shown in Fig. 3A, VII, a distinct peak of lankacidin C (1) appeared at 12 min. Production of lankacidin C in sample 1 was confirmed by ESI-MS. As shown in Fig. 3C, a molecular ion peak appeared at m/z 482 [MþNa]þ, which was two hydrogen atoms smaller than that of the substrate lankacidinol (4). In addition, all signals corresponding to lankacidin C (1) were observed in 1H-NMR spectrum of sample 1 (data not shown). On the other hand, no conversion was detected in the absence of PQQ (sample 2) as well as in the cell-free supernatant of S. lividans TK64/pHSA81 (sample 3) (Fig. 3A and C). These results revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety in lankacidinol leading to lankacidin C with the help of PQQ. Enzymatic properties of Orf23 SDS-PAGE analysis of the recombinant Orf23 protein is shown in Fig. S2. A 59-kDa band appeared in the purified protein fraction through Ni-NTA agarose affinity chromatography, however, several unclassified bands were observed. Use of TALON resin (charged with cobalt) resulted in no improvement of protein purity. The Orf23 protein purified by Ni2þ-affinity chromatography also showed the dehydrogenase activity in the presence of PQQ (Fig. S2), however, a large amount
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 2. Alignment of amino acid sequences among bacterial quinoprotein dehydrogenases including Orf23. Orf23 from S. rochei 7434AN4 (GenBank accession number: BAC76481); HR51_10690 from Burkholderia cepacia (KER73041); Sce5403 from Sorangium cellulosum So ce56 (CAN95566); PvaA from Pseudomonas sp. VM15C (BAA94193). Conserved amino acids are marked with asterisks (identical), colons (highly similar), and periods (weakly similar). The boxed sequences are the conserved W-motifs (W1-W8) those are widely found in quinoproteins/quinohemoproteins. Gray background indicates the two adjacent cysteine residues responsible for disulfide ring formation. Underline indicates the conserved heme c binding motif (CxxCH).
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 3. Analysis of metabolites produced by strain KK23 and enzymatic conversion by Orf23 protein. (A) HPLC chromatograms at 230 nm acquired using photodiode array detector. I, Strain 51252 (parent); II, strain KK23 (Dorf23); III, lankacidin C (1); and IV, lankacidinol (4). In vitro enzymatic conversion of 4 was performed in the cell-free extract of S. lividans TK64/pYY03 with exogeneous PQQ (sample 1; V), in the cell-free extract of S. lividans TK64/pYY03 without PQQ (sample 2; VI), and in the cell-free extract of S. lividans TK64/pHSA81 with exogeneous PQQ (sample 3; VII). The crude extracts or purified 1 and 4 were applied on a COSMOSIL Cholester column (4.6 x 250 mm, Nacalai Tesque) and eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 0.7 ml/min (B) TLC analysis of the crude extract of strains 51252 and KK23. TLC plate was developed with CHCl3/MeOH ¼ 15:1. (C) Electrospray-ionization mass spectra of samples 1e3 obtained by in vitro enzymatic conversion (see panel A).
of cell (50e100 times) was necessary to obtain the sufficient protein fraction. Due to a low quantity of the recombinant Orf23 protein, its biochemical analysis was carried out using a dialyzed cell-free supernatant without further purification. The pH dependency was first examined by using HEPES buffer in the pH range of 6.0e8.5. The dehydrogenase activity was calculated by the peak intensity ratios of lankacidin C to lankacidinol on the HPLC chromatogram. The dehydrogenase Orf23 was highly active around pH 6.5e7.5, with a maximum activity at pH 7.5. The relative activities at pH 6.5 and 7.0 were about 75% of the maximum activity. At pH 6.0, 8.0, and 8.5, the relative activities were 55%, 27%, and 12%, respectively. We then examined the substrate specificity of the Orf23 protein using two lankacidinol derivatives, lankacidinol A (2) and iso-
lankacidinol (3) (Fig. 1). Enzyme assays were carried out at pH 7.0 to minimize alkaline hydrolysis of the C-7 acetate in lankacidinol A. Although their conversion efficiencies were less than that of lankacidinol (4) (34% for lankacidinol A and 53% for iso-lankacidinol), both compounds were converted to the corresponding C-24 keto compounds. In vitro enzymatic conversion of lankacidinol A (2) in the orf23recombinant (sample 1) was analyzed and compared with the control recombinant (sample 2) (Fig. 4A and B). When eluted with acetonitrile/phosphate buffer (10 mM, pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml/min, a distinct peak (compound 5) appeared at 29.8 min in the orf23-recombinant (Fig. 4A, I), but not in the control recombinant (Fig. 4A, II). The ESI-MS analysis of sample 1 showed that a new parent ion peak appeared at m/z 524, two mass units smaller than
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 4. Substrate specificity of Orf23 protein. (A) HPLC chromatograms at 230 nm acquired using photodiode array detector. In vitro enzymatic conversion of lankacidinol A (2) was performed in the cell-free extract of S. lividans TK64/pYY03 with PQQ (sample 1; I), and in the cell-free extract of S. lividans TK64/pHSA81 with PQQ (sample 2; II). Purified 2 and 5 in MeOH are shown in items III and IV, respectively. These samples were eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml/min. (B) Electrospray-ionization mass spectra of samples 1 and 2 obtained by in vitro enzymatic conversion (see panel A). (C) HPLC chromatograms at 230 nm acquired using photodiode array detector. In vitro enzymatic conversion of iso-lankacidinol (3) was performed in the cell-free extract of S. lividans TK64/pYY03 with PQQ (sample 3; I), and in the cell-free extract of S. lividans TK64/pHSA81 with PQQ (sample 4; II). Purified 3 and 6 in MeOH are shown in items III and IV, respectively. These samples were applied on a COSMOSIL Cholester column (4.6 x 250 mm, Nacalai Tesque) and eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 0.7 ml/min. (D) Electrospray-ionization mass spectra of samples 3 and 4 obtained by in vitro enzymatic conversion (see panel C).
that of the substrate (Fig. 4B, I). Compound 5 was prepared by repeated enzymatic reactions, and purified to homogeneity. Its 1Hand 13C-NMR assignments are shown in Table S1. When compared with lankacidinol A (2), a doublet methyl proton H-25 (dH 1.36) was changed to a singlet methyl proton (dH 2.47). Other signals agreed with those of authentic lankacidin A (5). Lankacidin A (sedecamycin) is a 7-O-acetyl derivative of lankacidin C and exhibits a significant antimicrobial activity (2,22,23) (Table S1). In the case of iso-lankacidinol (3) (samples 3 and 4 in Fig. 4), a new peak (compound 6) appeared at 16.5 min in the orf23-
recombinant (Fig. 4C, I), not in the control recombinant (Fig. 4C, II). The ESI-MS spectrum of sample 4 showed the presence of a product with two mass units smaller than iso-lankacidinol (Fig. 4D, I), supporting dehydrogenation of the substrate. Compound 6 was also prepared through repeated enzymatic reactions and purified to homogeneity. Compared with iso-lankacidinol, a deshieled proton H-24 (dH 4.09) disappeared and a doublet methyl proton H-25 (dH 1.36) was changed to a singlet signal (dH 2.49). In the 13C-NMR, a methine carbon C-24 (dC 69.2) was changed to a quaternary carbon (dC 196.3). Other signals of compound 6 were almost identical to
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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TABLE 2. Effect of divalent metal cations on the dehydrogenase activity by Orf23. Additivea
Relative activityb
None Ca2þ Mn2þ Cu2þ Fe2þ Ni2þ Ba2þ Co2þ Mg2þ Zn2þ EDTA
22.3 100 80.0 50.0 46.1 41.4 33.4 30.3 27.1 25.3 0
a Concentrations of divalent metal cations were 0.6 mM. Concentration of EDTA was 1 mM. b Conversion ratio was calculated by peak intensity of lankacidinol (substrate) and lankacidin C (product) on HPLC chromatogram. Relative activity was defined as Ca2þ 100%.
those of iso-lankacidinol (Table S1). When compared with lankacidin C (1), the H-5 proton signal was upfield-shifted from dH 4.43 to dH 3.57, and the H-4 signal was downfield-shifted from dH 2.36e2.44 to dH 2.75. From these data, compound 6 was determined to be iso-lankacidin C (6) as a novel compound. Thus, Orf23 is the first example of the quinoprotein dehydrogenase involved in antibiotic biosynthesis. In general, quinoprotein dehydrogenases require a divalent metal ion such as Ca2þ to coordinate with a quinone cofactor at the active site residues (13). Hence, metal ion requirement for the dehydrogenase activity was calculated from the HPLC peak intensity of lankacidinol (substrate) and lankacidin C (product) in the presence of PQQ (13 mM). Among the divalent metals tested, Ca2þ led to a 4.5-fold enhancement of the dehydrogenase activity (Table 2). Other divalent metals including Mn2þ, Cu2þ, Fe2þ, Ni2þ also enhanced the dehydrogenase activity at 3.6, 2.2, 2.1, and 1.9-folds, respectively. Addition of EDTA completely inhibited the dehydrogenase activity. DISCUSSION In this work, we analyzed the function of the quinoprotein dehydrogenase (Orf23) coded on the linear plasmid pSLA2-L in lankacidin biosynthesis. In silico analysis of Orf23 by amino acid alignment (Fig. 2) indicated that Orf23 is classified into the PVA-DH family. The most characterized PVA-DH, PvaA, from Pseudomonas sp. VM15C carries a characteristic heme c binding motif and the two adjacent Cys residues (19). However, Orf23 contains neither of them, suggesting its different catalytic properties. Reinvestigation of the metabolites of the orf23 disruptant (KK23) showed that this mutant produced lankacidinol A not but lankacidin C. Total amount of lankacidin derivatives in strain KK23 (4.4 mg/L for compounds 2e4) was slightly smaller (61%) than that in the parent strain 51252 (7.2 mg/L for compounds 1e4). The function of the Orf23 protein has been finally confirmed by in vitro conversion of lankacidinol (4) to lankacidin C (1) (Fig. 1B). The lactate moiety (C23-C25) in lankacidinol was oxidized to the pyruvate moiety in lankacidin C by Orf23 at the final step of biosynthesis. This enzymatic activity agreed well with our previous observation that S. lividans recombinant harboring lkcA-lkcO accumulated lankacidinol A, not lankacidin C (8). This is the first report of quinoprotein involved in antibiotic biosynthesis. He et al. (11) studied the origin of the C3 unit (C23-C25) of lankacidin using the gene disruptants of orf19 and orf21. They speculated that this moiety is derived from glycerate, which is oxidized to pyruvate and then reduced to lactate, by analogy to the biosynthesis of the C3
7
unit of antibiotic FR901464. However, they did not isolate any biosynthetic intermediates from the disruptants, possibly because these reactions may occur at the early stage of biosynthesis. The Orf23 protein also converted lankacidinol A (2) and isolankacidinol (3) to lankacidin A (5) and iso-lankacidin C (6), respectively, although they were not detected in the wild-type strain under our laboratory condition. This finding may reflect that lankacidinol (4) acts as a preferred substrate for Orf23 in lankacidin biosynthesis in S. rochei. Orf23 was unable to oxidize the linear biosynthetic intermediates of lankacidin, LC-KA05 and its 7O-deacetoxy-7-hydroxy congener (LC-KA05-2), obtained from the disruptant of the amine oxidase (lkcE) (7), indicating the importance of the 17-membered macrocyclic structure for the dehydrogenase activity (Fig. S4). Orf23 was neither able to oxidize simpler molecules including D-glucose and lactate (data not shown). Exogenous addition of Ca2þ resulted in a 4.5-fold increase of the dehydrogenase activity. Other divalent metals including Mn2þ, Cu2þ, Fe2þ, Ni2þ also enhanced the dehydrogenase activity, while EDTA treatment completely inhibited, suggesting the importance of divalent metal ion for Orf23. Similarly, stimulating effect by Ca2þ and inhibitory effect by EDTA treatment on the dehydrogenase activity was reported for PvaA from Pseudomonas sp. VM15C (13). In the case of sorbosone dehydrogenase from Ketogulonicigenium vulgare, Cu2þ completely inhibited its activity, while no enhancement of the activity was observed by Ca2þ, Co2þ, Ni2þ, Zn2þ, or Mg2þ (26). In polypropylene glycol dehydrogenase from the bacterium Stenotrophomonas maltophilia, addition of Ca2þ, Mg2þ or EDTA did not affect the dehydrogenase activity (27). Thus, metal dependency is variable in quino(hemo)proteins. PQQ (Fig. S1) is the most characterized quinone cofactor in bacterial dehydrogenases. Under physiological conditions, PQQ readily reacts with various amino acids to form imidazolopyrroloquinoline (IPQ) derivatives (Fig. S1) (24). In addition, several types of quinone cofactors were reported in association with quino(hemo)protein dehydrogenases (25) (Fig. S1). We previously reported that the lkcL mutant (strain FS7) did not show any inhibitory zone on bioautography (7). However, extensive analysis here indicated that mutant FS7 does not produce lankacidinol A or lankacidin C, but produces a trace amount of lankacidinol (0.3 mg/L), the total amount of lankacidin derivatives being 4.2% of the parent (Fig. S3). Therefore, it is possible that quinone may have another (regulatory?) roles in lankacidin biosynthesis in addition to a cofactor for dehydrogenation of the lactate moiety to pyruvate. Recent genome sequencing analysis by next-generation sequencer indicated that a quinone cofactor/quino(hemo)protein pair is distributed in various gram-positive bacteria including Streptomyces species. Nineteen pqq clusters, including S. rochei linear plasmid pSLA2-L, have been annotated among 364 Streptomyces species currently available in the GenBank database (as of January 1, 2018). Extensive analysis of the pqq cluster together with quino(hemo)proteins may lead to unveil their physiological/biological functions in Streptomyces species and other gram-positive bacteria. Supplementary data related to this article can be found at https://doi.org/10.1016/j.jbiosc.2018.03.006.
ACKNOWLEDGMENTS We thank Profs. M. Kobayashi and Y. Hashimoto (Tsukuba University) for providing constitutive expression vector pHSA81. This work was supported by JSPS KAKENHI Grant Numbers JP16H04917, JP17H05446 (to K.A.), the JSPS A3 Foresight Program "Chemical & Synthetic Biology of Natural Products", and the JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (Grant Number S2902).
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J. BIOSCI. BIOENG., 14. Matsushita, K., Toyama, H., Yamada, M., and Adachi, O.: Quinoproteins: structure, function, and biotechnological applications, Appl. Microbiol. Biotechnol., 58, 13e22 (2002). 15. Toyama, H., Mathews, F. S., Adachi, O., and Matsushita, K.: Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology, Arch. Biochem. Biophys., 428, 10e21 (2004). 16. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A.: Practical Streptomyces genetics. The John Innes Foundation, Norwich, United Kingdom (2000). 17. Zhang, H., Shinkawa, H., Ishikawa, J., Kinashi, H., and Nimi, O.: Improvement of transformation system in Streptomyces using a modified regeneration medium, J. Ferment. Bioeng., 83, 217e221 (1997). 18. Suzuki, T., Mochizuki, S., Yamamoto, S., Arakawa, K., and Kinashi, H.: Regulation of lankamycin biosynthesis in Streptomyces rochei by two SARP genes, srrY and srrZ, Biosci. Biotechnol. Biochem., 74, 819e827 (2010). 19. Shimao, M., Tamogami, T., Nishi, K., and Harayama, S.: Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp. strain VM15C, Biosci. Biotechnol. Biochem., 60, 1056e1062 (1996). 20. Blake, C. C. F., Ghosh, M., Harlos, K., Avezoux, A., and Anthony, C.: The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues, Nat. Struct. Mol. Biol., 1, 102e105 (1994). 21. Afolabi, P. R., Mohammed, F., Amaratunga, K., Majekodunmi, O., Dales, S. L., Gill, R., Thompson, D., Cooper, J. B., Wood, S. P., Goodwin, P. M., and Anthony, C.: Site-directed mutagenesis and X-ray crystallography of the PQQcontaining quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome cL, Biochemistry, 40, 9799e9809 (2001). 22. Uramoto, M., Otake, N., Ogawa, Y., and Yonehara, H.: The structures of bundlin A (lankacidin) and bundlin B, Tetrahedron Lett., 2249e2254 (1969). 23. Harada, S., Kishi, T., and Mizuno, K.: Studies on T-2636 antibiotics. II Isolation and chemical properties of T-2636 antibiotics, J. Antibiot., 24, 13e22 (1971). 24. Urakami, T., Sugamura, K., and Niki, E.: Characterization of imidazopyrroloquinoline compounds synthesized from coenzyme PQQ and various amino acids, Biofactors, 5, 75e81 (1995-1996). 25. Klinman, J. P. and Bonnot, F.: Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ, Chem. Rev., 114, 4343e4365 (2014). 26. Miyazaki, T., Sugisawa, T., and Hishino, T.: Pyrroloquinoline quinonedependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid, Appl. Env. Microbiol., 72, 1487e1495 (2006). 27. Tachibana, S., Kuba, N., Kawai, F., Duine, J. A., and Yasuda, M.: Involvement of a quinoprotein (PQQ-containing) alcohol dehydrogenase in the degradation of polypropylene glycols by the bacterium Stenotrophomonas maltophilia, FEMS Microbiol. Lett., 218, 345e349 (2004).
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4 Yusuke Yamauchi, Yosi Nindita, Keisuke Hara, Asako Umeshiro, Yu Yabuuchi, Toshihiro Suzuki, Haruyasu Kinashi, and Kenji Arakawa* Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan Received 27 January 2018; accepted 9 March 2018 Available online xxx
Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and isolankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A ([sedecamycin) and isolankacidin C. Addition of various divalent metal cations, especially Ca2D, enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biosynthesis; Quinoprotein dehydrogenase; Secondary metabolite; Antibiotics; Pyrroloquinoline quinone]
A group of unique 17-membered carbocyclic polyketide antibiotics lankacidins (Fig. 1) are produced by Streptomyces rochei 7434AN4 that carries three linear plasmids, pSLA2-L, -M, and -S (1). Lankacidins show various biological activities including antimicrobial action against gram-positive bacteria (2). X-ray crystallographic analysis revealed that lankacidin C (1; Fig. 1) and lankamycin (14-membered macrolide antibiotic) inhibit peptide formation synergistically by binding to the neighboring sites in the large bacterial ribosomal subunit (3,4). In addition, lankacidin C enhances stable micro-tubulin assembly and displaces taxoids from their binding sites (5). Nucleotide sequencing and gene inactivation experiments of the largest linear plasmid pSLA2-L revealed that the lankacidin biosynthetic (lkc) gene cluster is located on pSLA2-L (210,614 bp) (6). The lkc cluster contains a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid gene lkcA, three multidomain type-I PKS genes (lkcC, lkcF, lkcG), a discrete dehydratase gene lkcB, a discrete acyltransferase gene lkcD, and an amine oxidase gene lkcE (7). Remarkably, the lkc cluster carries only five ketosynthase (KS) domains although eight condensation reactions of malonyl CoA are necessary, suggesting a modulariterative mixed polyketide biosynthesis of lankacidin (8,9). Heterologous expression of the lkcA-lkcO genes in Streptomyces lividans led to the production of lankacidinol A (2), suggesting that a set of lkc genes is sufficient to construct the lankacidin skeleton (8). Recently, Dickschat et al. (10) reported that an additional dehydratase activity coded on lkcC is required for lankacidin
* Corresponding author. Tel./fax: þ81 82 424 7767. E-mail address: [email protected] (K. Arakawa).
biosynthesis and He et al. (11) speculated the origin of C3 unit (C23C25) amide-bound to the lankacidin skeleton. One of the striking features of the lkc gene cluster is the presence of biosynthetic genes (lkcK-lkcO) for pyrroloquinoline quinone (PQQ; Fig. S1) at its left end (7). This was the first report of the PQQ cluster in gram-positive bacteria. PQQ, the most characterized quinone cofactor, is known as a third family of redox cofactor in various quinoprotein and quinohemoprotein dehydrogenases (12e15). These quino(hemo)proteins utilize quinone cofactor to catalyze dehydrogenation of primary or secondary hydroxyl groups in sugars and alcohols in the periplasm of gram-negative bacteria. Hence we focused on the PQQ biosynthetic cluster lkcK-O (orf8-4) and the quinoprotein dehydrogenase (orf23) gene coded on pSLA2L in S. rochei. Strain FS7, a disruptant of the lkcL (pqqC) gene, showed no inhibitory zone around Rf ¼ 0.5 (CHCl3-MeOH ¼ 15:1, v/v) on TLC bioautography when compared with the parent strain. Its deficiency was restored by exogenous addition of PQQ (7), indicating its crucial role in lankacidin biosynthesis. Nevertheless, a mutant of the orf23 gene showed a distinct inhibitory zone around Rf ¼ 0.5. Here we reinvestigated the metabolic profile of the orf23 disruptant, and describe an extensive biochemical characterization of the quinoprotein dehydrogenase protein involved in lankacidin biosynthesis in S. rochei 7434AN4.
MATERIALS AND METHODS Strains, reagents, and culture conditions All the strains and plasmids used in this study are listed in Table 1. S. rochei strain 51252 that carries only pSLA2-L was used as a parent strain (1). Strain KK23, the orf23 disruptant of strain 51252, was constructed previously (7). YM medium (0.4% yeast extract, 1.0% malt extract, and
1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 1. Structures of lankacidin antibiotics and dehydrogenation step in lankacidin biosynthesis. (A) Structures of lankacidin C (1), lankacidinol A (2), iso-lankacidinol (3), lankacidinol (4), lankacidin A (5), and iso-lankacidin C (6). Ac, acetyl; Me, methyl. (B) Dehydrogenation step catalyzed by Orf23 in lankacidin biosynthesis.
0.4% D-glucose, pH 7.3) was used for antibiotic production. For protoplast preparation and protein expression, S. lividans TK64 and its recombinants were cultured in YEME liquid medium (16). Protoplasts were regenerated on R1M solid medium (17). PQQ disodium salt was purchased from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan).
monitored at 230 nm with a JASCO MD-2010 multi-wavelength photodiode array detector. TLC was developed with CHCl3/MeOH (15:1, v/v) and baked after staining with anisaldehyde-H2SO4. High resolution ESI-MS spectra were measured by a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
Isolation of metabolites Strains were cultivated in YM liquid medium at 28 C for 2 days. The culture broth was extracted twice with equal volume of EtOAc. The combined organic phase was dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was passed through Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) with methanol. The fractions containing lankacidins were combined, and further purified by silica gel chromatography with two different solvent systems of CHCl3-methanol ¼ 50:1e10:1 (v/v) and toluene-EtOAc ¼ 1:3 (v/v). Spectral data including 1H and 13C NMR assignments for compounds 1e4 have already been reported (7,18) (Table S1).
Preparation of the recombinant Orf23 protein A 1.6-kb PCR fragment containing orf23 (nt 41,354-42,985 of pSLA2-L) was amplified using the template cosmid B10 (7) and two primers, 23fNde and 23rHind (Table 1). The PCR fragment was digested with NdeI and HindIII and cloned into pKAR3063H (18), a constitutive expression vector carrying N-terminal (His)6-tag sequence in pHSA81, to give pYY03. The S. lividans TK64 recombinant harboring pYY03 was grown at 28 C for 72 h in YEME liquid medium (34% sucrose) containing 10 mg/ml of thiostrepton. Streptomyces cultures were harvested by centrifugation, and the residual cells were resuspended in 50 mM phosphate buffer (pH 8.0) and disrupted by sonication for three cycles of 20-sec with 0.5-min intervals on ice. The cell-free supernatant was dialyzed when necessary.
Analysis of metabolites Metabolites were analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and electrospray ionization-mass spectrometry (ESI-MS). The crude extract was dissolved in acetonitrile, applied on a reverse-phase HPLC column (Cosmosil Cholester 4.6 x 250 mm; Nacalai Tesque, Kyoto, Japan), eluted with acetonitrile/phosphate buffer (10 mM, pH 8.2) (3:7, v/v) at a flow rate of 0.7e1.0 ml/min, and then
In vitro conversion of lankacidinol in the Orf23 recombinant A standard assay mixture (1 ml) contains 250 mM lankacidinol (4; Fig. 1), 13 mM PQQ, 0.6 mM CaCl2, and the cell-free supernatant of S. lividans TK64/pYY03 (200 ml; prepared from 20-ml culture broth) in 50 mM HEPES buffer (pH 7.5). After incubation at
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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QUINOPROTEIN DEHYDROGENASE IN LANKACIDIN BIOSYNTHESIS
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TABLE 1. Bacterial strains, plasmids, and oligonucleotides used in this study. Strains/plasmids/oligonucleotides
Properties/product
Strain S. rochei 7434AN4 51252 KK23 S. lividans TK64 TK64/pYY03 TK64/pHSA81 Plasmid SuperCos-1 cosmid B10 pHSA81 pKAR3063H pYY03 Designed oligonucleotide 23fNde 23rHind
Source/ref.
Wild type (pSLA2-L,M,S) pSLA2-L orf23::kan of pSLA2-L in strain 51252
1 1 7
pro-2, str-6 Strain TK64 with plasmid pYY03, tsr, (His)6-tagged orf23 Strain TK64 with plasmid pHSA81, tsr
16 This study This study
Cosmid vector, bla 45.4-kb pSLA2-L DNA (nt 3,341-48,756) cloned into SuperCos-1 at BamHI site Constitutive expression vector in Streptomyces, tsr Constitutive expression vector in Streptomyces, tsr, N-terminal (His)6-tag sequence 1.6 kb NdeI-HindIII PCR fragment carrying orf23 cloned into pKAR3063H
Stratagene 7 M. Kobayashi 18 This study
50 -CGACATATGAGCACCAGGACCACGCCTG-30 50 -ATTAAGCTTTCAGCGGCCGCGTCTGCAC-30
This study This study
37 C for 2 h, the mixture was extracted with equal volume of EtOAc. The organic phase was concentrated in vacuo and then analyzed by HPLC and ESI-MS. The cell-free supernatant of S. lividans TK64/pHSA81 was also used for negative control. To evaluate the importance of a quinone cofactor, the assay mixture was incubated in the absence of PQQ. Substrate specificity of Orf23 Substrate specificity was estimated by varying the substrate in the standard assay mixture above mentioned. Lankacidinol A (2) and iso-lankacidinol (3; Fig. 1) were used instead of 4 at a concentration of 250 mM. To avoid alkaline hydrolysis of 2, a series of enzymatic assay was carried out at pH 7.0. Metal requirement Metal ion requirement for Orf23 was analyzed by varying the divalent metal ion in the standard assay mixture. Instead of CaCl2, eight metal cations including MgCl2, MnCl2, FeSO4, NiCl2, CoCl2, Ba(OH)2, CuCl2, ZnCl2 were tested at a concentration of 0.6 mM. EDTA (1.0 mM) was also used for a negative control of divalent metal ion.
RESULTS Primary structure of Orf23 The Orf23 protein shows considerable similarities with HR51_10690 protein from Burkholderia cepacia RB-39 (56% identity, 70% similarity) and Sce5403 protein from Sorangium cellulosum So ce56 (43% identity, 56% similarity), both of which were classified as the polyvinyl alcohol dehydrogenase (PVA-DH) family. Amino acid sequences among these proteins together with the most-studied PVA-DH, PvaA, from Pseudomonas sp. VM15C (19) were aligned and compared (Fig. 2). All the proteins contain the anti-parallel b-sheet structures, called W-motifs, that were found in quino(hemo) proteins (13e15). Its consensus sequence, AxDxxxGK(E)xxW, is well conserved in W1, W2, W3, W4, W6, and W7 (Fig. 2). When compared with PvaA, however, Orf23 has neither two adjacent cysteine residues (located between W2 and W3) nor a heme cbinding motif (CxxCH) at the N-terminus. The former makes an eight-membered disulfide ring that causes a hydrophobic interaction with quinone cofactor (20). The latter motif is involved in the binding to heme c, which serves as an electron acceptor from the reduced PQQ prosthetic group (21). Lack of these signature motifs in Orf23 suggests a different catalytic action during dehydrogenation process. Metabolite profile in the orf23 disruptant In our previous study, the orf23 mutant (strain KK23) exhibited a distinct inhibitory zone around Rf ¼ 0.5 (CHCl3-MeOH ¼ 15:1, v/v) on TLC bioautography (7). We now reinvestigated the metabolite profile of strain KK23 by reverse phase HPLC (Fig. 3A). Four peaks with a characteristic UV absorbance at 230 nm (flow rate; 0.7 ml/min) were detected in the parent strain 51252 (Fig. 3A, I); lankacidinol (4) at 6.0 min, iso-lankacidinol (3) at 7.6 min, lankacidin C (1) at
12.1 min, and lankacidinol A (2) at 21.3 min. Whereas strain KK23 showed three peaks of compounds 2e4, but not that of 1 (Fig. 3A, II). The distinct structural difference between lankacidin C (1) and compounds 2e4 is the oxidation degree at C-24 position; ketone in lankacidin C (1) and hydroxyl in compounds 2e4. This finding notified us that Orf23 functions to convert the C23-C25 lactate moiety to pyruvate in lankacidin biosynthesis. Hence, the inhibitory zone detected in strain KK23 in our previous study was not caused by lankacidin C but was by lankacidinol A, whose Rf value was around 0.45 (Fig. 3B). Average yields of compounds 2e4 in strain KK23 were 1.5, 0.4, and 2.5 mg per liter, respectively, while those of compounds 1e4 in strain 51252 were 5.5, 0.6, 0.2, and 0.9 mg per liter, respectively. In vitro bioconversion by Orf23 To study the function of the Orf23 protein in lankacidin biosynthesis, we cloned orf23 into various E. coli expression vectors. However, no protein expression was observed although various culture conditions and vectors were tested. Hence, the orf23 gene was cloned into pKAR3063H (18), a derivative of streptomycete constitutive expression vector pHSA81 (Profs. M. Kobayashi and Y. Hashimoto, personal communication), to afford pYY03. In order to examine the dehydrogenase activity of Orf23, lankacidinol (4) was treated with the cell-free supernatant of S. lividans TK64/pYY03 in the presence of PQQ, and the assay mixture (sample 1) was analyzed by HPLC and ESI-MS. As shown in Fig. 3A, VII, a distinct peak of lankacidin C (1) appeared at 12 min. Production of lankacidin C in sample 1 was confirmed by ESI-MS. As shown in Fig. 3C, a molecular ion peak appeared at m/z 482 [MþNa]þ, which was two hydrogen atoms smaller than that of the substrate lankacidinol (4). In addition, all signals corresponding to lankacidin C (1) were observed in 1H-NMR spectrum of sample 1 (data not shown). On the other hand, no conversion was detected in the absence of PQQ (sample 2) as well as in the cell-free supernatant of S. lividans TK64/pHSA81 (sample 3) (Fig. 3A and C). These results revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety in lankacidinol leading to lankacidin C with the help of PQQ. Enzymatic properties of Orf23 SDS-PAGE analysis of the recombinant Orf23 protein is shown in Fig. S2. A 59-kDa band appeared in the purified protein fraction through Ni-NTA agarose affinity chromatography, however, several unclassified bands were observed. Use of TALON resin (charged with cobalt) resulted in no improvement of protein purity. The Orf23 protein purified by Ni2þ-affinity chromatography also showed the dehydrogenase activity in the presence of PQQ (Fig. S2), however, a large amount
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 2. Alignment of amino acid sequences among bacterial quinoprotein dehydrogenases including Orf23. Orf23 from S. rochei 7434AN4 (GenBank accession number: BAC76481); HR51_10690 from Burkholderia cepacia (KER73041); Sce5403 from Sorangium cellulosum So ce56 (CAN95566); PvaA from Pseudomonas sp. VM15C (BAA94193). Conserved amino acids are marked with asterisks (identical), colons (highly similar), and periods (weakly similar). The boxed sequences are the conserved W-motifs (W1-W8) those are widely found in quinoproteins/quinohemoproteins. Gray background indicates the two adjacent cysteine residues responsible for disulfide ring formation. Underline indicates the conserved heme c binding motif (CxxCH).
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FIG. 3. Analysis of metabolites produced by strain KK23 and enzymatic conversion by Orf23 protein. (A) HPLC chromatograms at 230 nm acquired using photodiode array detector. I, Strain 51252 (parent); II, strain KK23 (Dorf23); III, lankacidin C (1); and IV, lankacidinol (4). In vitro enzymatic conversion of 4 was performed in the cell-free extract of S. lividans TK64/pYY03 with exogeneous PQQ (sample 1; V), in the cell-free extract of S. lividans TK64/pYY03 without PQQ (sample 2; VI), and in the cell-free extract of S. lividans TK64/pHSA81 with exogeneous PQQ (sample 3; VII). The crude extracts or purified 1 and 4 were applied on a COSMOSIL Cholester column (4.6 x 250 mm, Nacalai Tesque) and eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 0.7 ml/min (B) TLC analysis of the crude extract of strains 51252 and KK23. TLC plate was developed with CHCl3/MeOH ¼ 15:1. (C) Electrospray-ionization mass spectra of samples 1e3 obtained by in vitro enzymatic conversion (see panel A).
of cell (50e100 times) was necessary to obtain the sufficient protein fraction. Due to a low quantity of the recombinant Orf23 protein, its biochemical analysis was carried out using a dialyzed cell-free supernatant without further purification. The pH dependency was first examined by using HEPES buffer in the pH range of 6.0e8.5. The dehydrogenase activity was calculated by the peak intensity ratios of lankacidin C to lankacidinol on the HPLC chromatogram. The dehydrogenase Orf23 was highly active around pH 6.5e7.5, with a maximum activity at pH 7.5. The relative activities at pH 6.5 and 7.0 were about 75% of the maximum activity. At pH 6.0, 8.0, and 8.5, the relative activities were 55%, 27%, and 12%, respectively. We then examined the substrate specificity of the Orf23 protein using two lankacidinol derivatives, lankacidinol A (2) and iso-
lankacidinol (3) (Fig. 1). Enzyme assays were carried out at pH 7.0 to minimize alkaline hydrolysis of the C-7 acetate in lankacidinol A. Although their conversion efficiencies were less than that of lankacidinol (4) (34% for lankacidinol A and 53% for iso-lankacidinol), both compounds were converted to the corresponding C-24 keto compounds. In vitro enzymatic conversion of lankacidinol A (2) in the orf23recombinant (sample 1) was analyzed and compared with the control recombinant (sample 2) (Fig. 4A and B). When eluted with acetonitrile/phosphate buffer (10 mM, pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml/min, a distinct peak (compound 5) appeared at 29.8 min in the orf23-recombinant (Fig. 4A, I), but not in the control recombinant (Fig. 4A, II). The ESI-MS analysis of sample 1 showed that a new parent ion peak appeared at m/z 524, two mass units smaller than
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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FIG. 4. Substrate specificity of Orf23 protein. (A) HPLC chromatograms at 230 nm acquired using photodiode array detector. In vitro enzymatic conversion of lankacidinol A (2) was performed in the cell-free extract of S. lividans TK64/pYY03 with PQQ (sample 1; I), and in the cell-free extract of S. lividans TK64/pHSA81 with PQQ (sample 2; II). Purified 2 and 5 in MeOH are shown in items III and IV, respectively. These samples were eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml/min. (B) Electrospray-ionization mass spectra of samples 1 and 2 obtained by in vitro enzymatic conversion (see panel A). (C) HPLC chromatograms at 230 nm acquired using photodiode array detector. In vitro enzymatic conversion of iso-lankacidinol (3) was performed in the cell-free extract of S. lividans TK64/pYY03 with PQQ (sample 3; I), and in the cell-free extract of S. lividans TK64/pHSA81 with PQQ (sample 4; II). Purified 3 and 6 in MeOH are shown in items III and IV, respectively. These samples were applied on a COSMOSIL Cholester column (4.6 x 250 mm, Nacalai Tesque) and eluted with a mixture of acetonitrile-10 mM sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 0.7 ml/min. (D) Electrospray-ionization mass spectra of samples 3 and 4 obtained by in vitro enzymatic conversion (see panel C).
that of the substrate (Fig. 4B, I). Compound 5 was prepared by repeated enzymatic reactions, and purified to homogeneity. Its 1Hand 13C-NMR assignments are shown in Table S1. When compared with lankacidinol A (2), a doublet methyl proton H-25 (dH 1.36) was changed to a singlet methyl proton (dH 2.47). Other signals agreed with those of authentic lankacidin A (5). Lankacidin A (sedecamycin) is a 7-O-acetyl derivative of lankacidin C and exhibits a significant antimicrobial activity (2,22,23) (Table S1). In the case of iso-lankacidinol (3) (samples 3 and 4 in Fig. 4), a new peak (compound 6) appeared at 16.5 min in the orf23-
recombinant (Fig. 4C, I), not in the control recombinant (Fig. 4C, II). The ESI-MS spectrum of sample 4 showed the presence of a product with two mass units smaller than iso-lankacidinol (Fig. 4D, I), supporting dehydrogenation of the substrate. Compound 6 was also prepared through repeated enzymatic reactions and purified to homogeneity. Compared with iso-lankacidinol, a deshieled proton H-24 (dH 4.09) disappeared and a doublet methyl proton H-25 (dH 1.36) was changed to a singlet signal (dH 2.49). In the 13C-NMR, a methine carbon C-24 (dC 69.2) was changed to a quaternary carbon (dC 196.3). Other signals of compound 6 were almost identical to
Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006
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TABLE 2. Effect of divalent metal cations on the dehydrogenase activity by Orf23. Additivea
Relative activityb
None Ca2þ Mn2þ Cu2þ Fe2þ Ni2þ Ba2þ Co2þ Mg2þ Zn2þ EDTA
22.3 100 80.0 50.0 46.1 41.4 33.4 30.3 27.1 25.3 0
a Concentrations of divalent metal cations were 0.6 mM. Concentration of EDTA was 1 mM. b Conversion ratio was calculated by peak intensity of lankacidinol (substrate) and lankacidin C (product) on HPLC chromatogram. Relative activity was defined as Ca2þ 100%.
those of iso-lankacidinol (Table S1). When compared with lankacidin C (1), the H-5 proton signal was upfield-shifted from dH 4.43 to dH 3.57, and the H-4 signal was downfield-shifted from dH 2.36e2.44 to dH 2.75. From these data, compound 6 was determined to be iso-lankacidin C (6) as a novel compound. Thus, Orf23 is the first example of the quinoprotein dehydrogenase involved in antibiotic biosynthesis. In general, quinoprotein dehydrogenases require a divalent metal ion such as Ca2þ to coordinate with a quinone cofactor at the active site residues (13). Hence, metal ion requirement for the dehydrogenase activity was calculated from the HPLC peak intensity of lankacidinol (substrate) and lankacidin C (product) in the presence of PQQ (13 mM). Among the divalent metals tested, Ca2þ led to a 4.5-fold enhancement of the dehydrogenase activity (Table 2). Other divalent metals including Mn2þ, Cu2þ, Fe2þ, Ni2þ also enhanced the dehydrogenase activity at 3.6, 2.2, 2.1, and 1.9-folds, respectively. Addition of EDTA completely inhibited the dehydrogenase activity. DISCUSSION In this work, we analyzed the function of the quinoprotein dehydrogenase (Orf23) coded on the linear plasmid pSLA2-L in lankacidin biosynthesis. In silico analysis of Orf23 by amino acid alignment (Fig. 2) indicated that Orf23 is classified into the PVA-DH family. The most characterized PVA-DH, PvaA, from Pseudomonas sp. VM15C carries a characteristic heme c binding motif and the two adjacent Cys residues (19). However, Orf23 contains neither of them, suggesting its different catalytic properties. Reinvestigation of the metabolites of the orf23 disruptant (KK23) showed that this mutant produced lankacidinol A not but lankacidin C. Total amount of lankacidin derivatives in strain KK23 (4.4 mg/L for compounds 2e4) was slightly smaller (61%) than that in the parent strain 51252 (7.2 mg/L for compounds 1e4). The function of the Orf23 protein has been finally confirmed by in vitro conversion of lankacidinol (4) to lankacidin C (1) (Fig. 1B). The lactate moiety (C23-C25) in lankacidinol was oxidized to the pyruvate moiety in lankacidin C by Orf23 at the final step of biosynthesis. This enzymatic activity agreed well with our previous observation that S. lividans recombinant harboring lkcA-lkcO accumulated lankacidinol A, not lankacidin C (8). This is the first report of quinoprotein involved in antibiotic biosynthesis. He et al. (11) studied the origin of the C3 unit (C23-C25) of lankacidin using the gene disruptants of orf19 and orf21. They speculated that this moiety is derived from glycerate, which is oxidized to pyruvate and then reduced to lactate, by analogy to the biosynthesis of the C3
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unit of antibiotic FR901464. However, they did not isolate any biosynthetic intermediates from the disruptants, possibly because these reactions may occur at the early stage of biosynthesis. The Orf23 protein also converted lankacidinol A (2) and isolankacidinol (3) to lankacidin A (5) and iso-lankacidin C (6), respectively, although they were not detected in the wild-type strain under our laboratory condition. This finding may reflect that lankacidinol (4) acts as a preferred substrate for Orf23 in lankacidin biosynthesis in S. rochei. Orf23 was unable to oxidize the linear biosynthetic intermediates of lankacidin, LC-KA05 and its 7O-deacetoxy-7-hydroxy congener (LC-KA05-2), obtained from the disruptant of the amine oxidase (lkcE) (7), indicating the importance of the 17-membered macrocyclic structure for the dehydrogenase activity (Fig. S4). Orf23 was neither able to oxidize simpler molecules including D-glucose and lactate (data not shown). Exogenous addition of Ca2þ resulted in a 4.5-fold increase of the dehydrogenase activity. Other divalent metals including Mn2þ, Cu2þ, Fe2þ, Ni2þ also enhanced the dehydrogenase activity, while EDTA treatment completely inhibited, suggesting the importance of divalent metal ion for Orf23. Similarly, stimulating effect by Ca2þ and inhibitory effect by EDTA treatment on the dehydrogenase activity was reported for PvaA from Pseudomonas sp. VM15C (13). In the case of sorbosone dehydrogenase from Ketogulonicigenium vulgare, Cu2þ completely inhibited its activity, while no enhancement of the activity was observed by Ca2þ, Co2þ, Ni2þ, Zn2þ, or Mg2þ (26). In polypropylene glycol dehydrogenase from the bacterium Stenotrophomonas maltophilia, addition of Ca2þ, Mg2þ or EDTA did not affect the dehydrogenase activity (27). Thus, metal dependency is variable in quino(hemo)proteins. PQQ (Fig. S1) is the most characterized quinone cofactor in bacterial dehydrogenases. Under physiological conditions, PQQ readily reacts with various amino acids to form imidazolopyrroloquinoline (IPQ) derivatives (Fig. S1) (24). In addition, several types of quinone cofactors were reported in association with quino(hemo)protein dehydrogenases (25) (Fig. S1). We previously reported that the lkcL mutant (strain FS7) did not show any inhibitory zone on bioautography (7). However, extensive analysis here indicated that mutant FS7 does not produce lankacidinol A or lankacidin C, but produces a trace amount of lankacidinol (0.3 mg/L), the total amount of lankacidin derivatives being 4.2% of the parent (Fig. S3). Therefore, it is possible that quinone may have another (regulatory?) roles in lankacidin biosynthesis in addition to a cofactor for dehydrogenation of the lactate moiety to pyruvate. Recent genome sequencing analysis by next-generation sequencer indicated that a quinone cofactor/quino(hemo)protein pair is distributed in various gram-positive bacteria including Streptomyces species. Nineteen pqq clusters, including S. rochei linear plasmid pSLA2-L, have been annotated among 364 Streptomyces species currently available in the GenBank database (as of January 1, 2018). Extensive analysis of the pqq cluster together with quino(hemo)proteins may lead to unveil their physiological/biological functions in Streptomyces species and other gram-positive bacteria. Supplementary data related to this article can be found at https://doi.org/10.1016/j.jbiosc.2018.03.006.
ACKNOWLEDGMENTS We thank Profs. M. Kobayashi and Y. Hashimoto (Tsukuba University) for providing constitutive expression vector pHSA81. This work was supported by JSPS KAKENHI Grant Numbers JP16H04917, JP17H05446 (to K.A.), the JSPS A3 Foresight Program "Chemical & Synthetic Biology of Natural Products", and the JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (Grant Number S2902).
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Please cite this article in press as: Yamauchi, Y., et al., Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4, J. Biosci. Bioeng., (2018), https://doi.org/10.1016/j.jbiosc.2018.03.006