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1
Biosynthesis of 2’-chloropentostatin and 2’-amino-2’-deoxyadenosine 1
highlights a single gene cluster performing two independent pathways 2
in Actinomadura sp. ATCC 39365 3
Yaojie Gao,†,§ Gudan Xu,†,§ Pan Wu,†,§ Jin Liu,† You-sheng Cai, † Zixin Deng,† Wenqing Chen†,* 4 †Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and 5
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China 6 §Co-first author 7 *For Correspondence: Wenqing Chen, School of Pharmaceutical Sciences, Wuhan University, 8
Wuhan 430071, China. E-mail: [email protected], Tel: +86-27-68756713, Fax: +86-27-9
68759850. 10
Running Head: Biosynthesis of 2’-chloropentostatin 11
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AEM Accepted Manuscript Posted Online 3 March 2017Appl. Environ. Microbiol. doi:10.1128/AEM.00078-17Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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Abstract 26
2’-chloropentostatin (2’-Cl PTN, 2’-chloro-2’-deoxycoformycin) and 2’-amino-2’-27
deoxyadenosine (2’-Amino dA) are two adenosine-derived nucleoside antibiotics co-28
produced by Actinomadura sp. ATCC 39365. 2’-Cl PTN is a potent adenosine 29
deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring as well as a 30
chlorination at C-2’ of ribose, and 2’-Amino dA is an adenosine analog showing 31
bioactivity against RNA-type virus infection. However, the biosynthetic logic of them 32
has remained poorly understood. Here, we report the identification of a single gene 33
cluster (ada) essential for the biosynthesis of 2’-Cl PTN and 2’-Amino dA. Further 34
systematic genetic investigations suggest that 2’-Cl PTN and 2’-Amino dA are 35
biosynthesized by independent pathways. Moreover, we provide evidence that a 36
predicted cation/H+ antiporter AdaE is involved in the chlorination step during 2’-Cl 37
PTN biosynthesis. Notably, we demonstrate that 2’-Amino dA biosynthesis is initiated 38
by a NUDIX hydrolase AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that 39
the host ADA (designated as ADA1), capable of converting adenosine/2’-Amino dA to 40
inosine/2’-Amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. 41
These findings provide basis for the further rational pathway engineering of 2’-Cl PTN 42
and 2’-Amino dA production. 43
44
Importance 45
2’-Cl PTN/PTN and 2’-Amino dA have captivated the great interests of scientists 46
owing to their unusual chemical structures as well as remarkable bioactivities. 47
However, the precise logic for their biosynthesis has been elusive for decades. 48
3
Actually, identification and elucidation of their biosynthetic pathways not only 49
enriches the biochemical repertoire of novel enzymatic reactions, but may also lays 50
solid foundations for the pathway engineering and combinatorial biosynthesis of this 51
family of purine nucleoside antibiotics to generate novel hybrid analogs with 52
improved features. 53
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Key words: 2’-chloropentostatin, 2’-amino-2’-deoxyadenosine, nucleoside 55
antibiotics, biosynthesis, NUDIX hydrolase, adenosine deaminase 56
57
INTRODUCTION 58
Nucleoside antibiotics are a large family of important microbial natural products 59
harboring wide-range biological properties as well as distinctive structural features 60
(1-3). Their biosynthesis generally follows a succinct logic by sequential enzymatic 61
modification of nucleoside or nucleotide originating from primary metabolisms (1). 62
Usually, nucleosides and nucleotides play pleiotropic roles in most fundamental 63
cellular metabolisms, and therefore, nucleoside antibiotics are able to target the 64
biosyntheses of diverse biomacromolecules including nucleic acid, protein, and 65
glycan (1). 66
2’-chloropentostatin (2’-Cl PTN, 2’-chloro-2’-deoxycoformycin) and 2’-amino-2’-67
deoxyadenosine (2’-Amino dA) (Fig. 1A) are both purine-derived nucleoside 68
antibiotics concomitantly produced by Actinomudura sp. ATCC 39365 (4), and yet 69
they were reported to be individually produced as well by other Actinomycetes 70
strains (5, 6). Remarkably, the co-production phenomenon of the antibiotic 2’-Cl PTN 71
4
and 2’-amino dA has also been reported in the past decades for other antibiotics 72
pairs, including pentostatin (PTN)/arabinofuranosyladenine (Ara-A) (7), and 73
coformycin/formycin (Fig. 1A) (8). Mechanistically, 2’-Cl PTN mimics the transition-74
state intermediate of the adenosine deaminase (ADA) catalyzed reaction, and thus it 75
is a powerful irreversible ADA inhibitor with Ki=1.1×10-11 M (9). 2’-Cl PTN represents 76
a new group of nucleoside antibiotics with utility in the treatment of hematological 77
cancers (4), while it is not active against the indicator fungi and bacteria in the 78
presence of the test conc. 1 mg/ml (6). Moreover, 2’-Cl PTN is able to greatly 79
potentiate the antiviral efficacy of Ara-A, and the acute toxicity of 2’-Cl PTN in mice 80
was less than that of coformycin and PTN (6). Regarding 2’-Amino dA, it has been 81
shown to be selectively effective inhibitor against the replication of some riboviruses, 82
including the measles virus (10, 11). Very interestingly, two other related adenosine 83
analogs designated as 2’-amino-2'-deoxyguanosine (2’-Amino dG) (12) and 3’-amino-84
3’-deoxyadenosine (3’-Amino dA)(13) have been discovered prior to 2’-Amino dA (Fig. 85
1B). 2’-Amino dG indicates antibacterial activity against E. coli and antitumor activity 86
against sarcoma cells (12), and 3’-Amino dA shows significant antitumor activity 87
against ascitic tumors of mice (13). 88
Structurally, 2’-Cl PTN share an identical 1,3-diazepine ring with other related 89
antibiotics including PTN and coformycin, but contains an unusual chlorination at C-90
2’ position (4), and 2’-Amino dA is an adenosine analog featuring a C-2’ amino group 91
substituted for the corresponding hydroxyl group of adenosine (4). Previous 92
metabolic labeling experiments indicated that adenosine acts as the direct precursor 93
for the biosynthesis of both antibiotics (4). In addition, the C-7 origin of the “fat” 1,3-94
diazepine ring comes from C-1 of D-ribose by inserting the N-1 and C-6 of the intact 95
5
purine ring (7). In our recent report, we have revealed that a single gene cluster 96
performs two independent pathways during PTN and Ara-A biosynthesis, and further 97
demonstrated that their biosynthesis employs a protector-protege strategy with the 98
former capable of protecting the latter from deamination by the host adenosine 99
deaminase (14). Notably, 2’-Cl PTN has been reported as the first naturally-occurring 100
nucleoside natural product bearing the chloro group (15). As for the biosynthetic 101
origin of the chlorination, metabolic labeling experiments indicated that the 102
inorganic chloride in the medium could be utilized by Actinomuduan sp. ATCC 39365 103
serving as the chloro group in 2’-Cl PTN (4), however, the precise mechanism on how 104
the chlorination occurs during the 2’-Cl PTN biosynthesis has remained elusive for 105
decades. 106
In the present report, we reveal that 2’-Cl PTN and 2’-Amino dA biosynthesis 107
exploits a “a single gene cluster encoding two independent pathways” strategy, and 108
further demonstrate that the host adenosine deaminase ADA1 contributes to the 109
deamination of 2’-Amino dA to 2’-Amino deoxyinosine (2’-Amino dI) (Fig. 1B). 110
Moreover, we illustrate that a NUDIX hydrolase (AdaJ) governs the initial step of 2’-111
Amino dA biosynthesis. The deciphering of the biosynthetic puzzles for 2’-Cl PTN and 112
2’-Amino dA lays a solid foundation for the rational generation of designer 2’-Cl PTN 113
and 2’-Amino dA analogs via synthetic biology strategies, and undoubtedly expands 114
the chemical diversities concerning the biosynthesis of nucleoside natural products. 115
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MATERIALS AND METHODS 117
Strains, plasmids, primers, Enzymes, chemicals and general methods 118
6
Strains, plasmids used in this study are described in Table 1, and the relevant PCR 119
primers are listed in Table S1. All of the restriction enzymes and other enzymes used 120
in this study were purchased from New England Biolabs. The standards including 2’-121
Amino dA, 2’-Amino dI, and PTN were purchased from Aladdin Biotech, Hongene 122
biotech, and Shanghai Qifa Biotech, respectively. All of other chemicals were 123
purchased from Sigma-Aldrich, Thermo Scientific, or J&K Scientific. Standard 124
protocols used to manipulate E. coli or Streptomyces were based on those of Green 125
et al (16) or Kieser et al (17). 126
127
Sequencing and annotation of the genome of Actinomaduara sp. ATCC 39365 128
Genomic DNA of Actinomadura sp. ATCC 39365 was prepared according to the 129
standard protocol (17), and the genome sequencing was performed using the 130
Illumina HiseqTM2500 sequencing system, and the sequence data was then 131
assembled using Velvet software, and annotated using the Glimmer 3.0 software. 132
The online programs FramePlot 4.0beta (http://nocardia.nih.go.jp/fp4/) and 2ndFind 133
(http://biosyn.nih.go.jp/2ndfind/) were exploited for the accurate analysis of the 2’-Cl 134
PTN and 2’-Amino dA gene cluster. 135
136
Construction of the Actinomadura sp. LG1 mutant 137
For the construction of LG1 mutant, the left arm (1.9-kb) and right arm (2.5-kb) 138
were amplified with the primer pairs: JKLM LarmF/LarmR and JKLM RarmF/RarmR. 139
Afterwards, the left arm was digested with XbaI and BglII and cloned into the 140
corresponding sites of pOJ446 to produce pLG001. Later, the right arm, cleaved by 141
BglII, was cloned into the BglII-HpaI site of pLG001 to generate pLG002, and then the 142
7
kanamycin resistance gene (neo) was cloned into the BglII site of pLG002 to form 143
pLG003, which was subsequently introduced by conjugation into Actinomadura sp. 144
ATCC 39365. After that, the standard methods were conducted in the screening of 145
LG1 mutant (2). 146
147
Production, purification, and LC-MS analysis of related antibiotics 148
Production of related antibiotics by Actinomadura sp. ATCC 39365 was according 149
to the methods by Tunac et al (9). After fermentation, the broth was centrifuged at 150
8000 rpm for 10 minutes, and the supernatant was extracted with equal volume of n-151
butyl alcohol for three times. After concentration, the residue was redissolved in 152
water for further analysis. The LC-MS analysis of 2’-Cl PTN and 2’-Amino dA was 153
performed on a Thermo LTQ-Obitrap ESI HRMS machine equipped with a C-18 154
reversed-phase column (GL sciences, 5 µm, 4.6×250 mm) in an elution gradient of 155
5%-30% Methanol:0.15% TFA over 30 min at 0.5 ml/min, and the elution was 156
monitored at 254 nm with a DAD detector. 157
158
Genomic library construction and screening for Actinomadura sp. ATCC 39365 159
For the construction of pJTU2463b-derived genomic library for Actinomadura sp. 160
ATCC 39365, standard method was performed, using the EPI300-T1R as suitable host 161
cells, and the narrow-down PCR screening strategy (39365cluster-idF/R, 2463b-1F/1R, 162
2F/2R) was employed (3) to screen the positive cosmid 3G12 from the genomic 163
library. 164
165
In-frame deletion of the target ada genes by PCR-targeting strategy 166
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For targeted inactivation of the genes in ada gene cluser, a kanamycin resistance 167
cassette (neo) was amplified using corresponding primers 39365pcrtgtF/R (Table S1), 168
and then recombined into the target gene in 3G12 by PCR-targeting strategy to give 169
3G12::neo (18). The neo cassette was then deleted to produce a series of 3G12/∆ada 170
derivatives (Fig. S4) (19). The unmarked deletions were subsequently confirmed by 171
PCR using related pair of primers (Table S1). 172
173
Expression and purification of AdaJ and ADA1 in E. coli Rosetta(DE3)/pLysS 174
Using the primers listed in Table S1, pSJ8/adaJ and pET28a /ada1 were constructed 175
and subsequently transformed into E. coli Rosetta(DE3)/pLysS cells according to the 176
standard protocols (16). For adaJ specifically, its codon was optimized in advance 177
based on E. coli codon usage (See Supplemental data). Expression and purification for 178
both His6-tagged proteins were employed according to the method by Wu et al (14). 179
For AdaJ, it was expressed with a fusion MBP tag, after digestion by TEV, the target 180
purified protein proteins were then concentrated and stored with protein stock 181
buffer (25 mM Tris, pH 8.0, 150 mM NaCl, and 10% glycerol) using Amicon Ultra 182
filters. 183
184
Biochemical and relative activity assays of AdaJ 185
For AdaJ activity assay, the reaction mixture consisting of 50 mM TrisCl buffer 186
(pH7.5), 1 mM ATP, 100 mM KCl, 50 mM divalent ion (Mg2+, Co2+, Mn2+, Zn2+, Fe2+, 187
Cu2+) and 20 μg AdaJ was performed at 30°C for 4 h, then terminated by the addition 188
of equivalent volume methanol immediately. Following centrifugation to remove 189
protein, products were subsequently analyzed by HPLC (Shimadzu LC-20A) and LC-190
9
HRMS (Thermo LTQ-Obitrap). Equipped with a reverse phase C18 column (Inertsil 191
ODS-3, 4.6 × 250 mm, 5 μm), monitored by a DAD detector at 260 nm, HPLC was 192
performed at a flow rate of 0.5 ml/min with the elution gradient of 5%-20% 193
methanol:10 mM TEAA (pH7.0) over 20 min, then turn to the ratio of 5% methanol: 194
10 mM TEAA at 22 min, and continued till 30 min. LC-HRMS/MS was operated in an 195
ESI-ion trap mass spectrometer under the positive ion mode with drying gas 275°C, 196
10 L/ml and nebulizer pressure 30 psi. The activity assays were performed with 197
general protocol of EnzCheK Pyrophosphate Assay Kit (Thermo). 198
199
Accession numbers 200
The DNA sequence of the ada gene cluster is available in the GenBank database 201
under accession number KPO25768 and KY373246. 202
203
RESULTS 204
Reinvestigation of the target nucleoside metabolites produced by Actinomadura sp. 205
ATCC 39365 206
Actinomadura sp. ATCC 39365 has been previously characterized as a 2’-Cl PTN and 207
2’-Amino dA producer (4), but this strain has never been reported to produce other 208
related nucleoside antibiotics, including PTN and 2’-Amino dI, which are most likely 209
to be biosynthesized by this strain due to the fact that 2’-Cl PTN potentially originates 210
from PTN by direct chlorination, and 2’-Amino dA is prone to deamination by host 211
ADA to 2’-Amino dI. To address this question, we reinvestigated the metabolite 212
profiles of the sample of Actinomadura sp. ATCC 39365 by LC-MS analysis, and the 213
10
results indicated that it could generate the obvious characteristic [M+H]+ ion at 214
m/z=268.1021 and fragment ions at 136.9485 and 251.0468, in full agreement with 215
those of the 2’-Amino dI authentic standard (Fig. S1A-C), and the sample could also 216
produce distinctive [M+H]+ ion at m/z=269.1224 as well as fragment series 152.9804, 217
134.8981, and 251.0840, which well correspond to those of the PTN authentic 218
standard (Fig. 2B, Fig. S2A-B). Moreover, as anticipated, LC-MS results showed that 219
the sample of Actinomadura sp. ATCC 39365 could give apparent distinctive [M+H]+ 220
ions of 2’-Amino dA and 2’-Cl PTN (Fig. 2B-C, Fig. S2C-E), confirming the identity of 221
the strain as a 2’-Amino dA and 2’-Cl PTN producer as previously documented. These 222
data unquestionably demonstrated that Actinomadura sp. ATCC 39365 is also a 2’-223
Amino dI and PTN producer. 224
225
Identification of 2’-Cl PTN and 2’-Amino dA biosynthetic gene cluster 226
For identifying the target gene cluster responsible for 2’-Cl PTN and 2’-Amino dA 227
biosynthesis, the genome of Actinomadura sp. ATCC 39365 was sequenced by the 228
Illumina method, which rendered ca. 11.2-Mb non-redundant bases after assembly 229
of clean reads. The genome data of Actinomadura sp. ATCC 39365 was subsequently 230
annotated by Glimmer 3.0 software yielding 10,533 valid open reading frames 231
(ORFs). As both 2’-Cl PTN and PTN share the 1,3-diazepine scaffold, implicating that 232
they harbor the same biosynthetic logic, we thus use the three key enzymes 233
including PenA (ATP phosphoribosyltransferase), PenB (short-chain dehydrogenase), 234
and PenC (saicar synthetase) (19), in PTN biosynthetic pathway as probes to conduct 235
individual BlastP analysis against the genome of Actinomadura sp. ATCC 39365, 236
which leads to the location of the only one candidate gene cluster encoding enzymes 237
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involving ORF02747 (designated as AdaC, 58% identity to PenA), ORF02756 (AdaL, 238
53% identity to PenA), ORF02748 (AdaB, 70% identity to PenB), and ORF02749 (AdaA, 239
72% identity to PenC) (Table 2). This bioinformatics analysis result strongly suggests 240
that the target gene cluster is most likely involved in 2’-Cl PTN and PTN biosynthesis 241
in Actinomadura sp. ATCC 39365 (Fig. 2A). 242
Further examination of the surrounding region of adaABC results in the revealing 243
of the genes coding for an aminotransferase (AdaF) and a dehydrogenase (AdaG), 244
which are fully consistent with the predicted enzymes required for 2’-Amino dA 245
biosynthesis. According to the in silico analysis, adaFGHIJ constitute a transcriptional 246
unit (AdaJ is an annotated NUDIX hydrolase), and downstream are the three genes 247
individually encoding a phosphoribosyl isomerase A (AdaK, HisA homolog), an ATP 248
phosphoribosyltransferase (AdaL, HisG homolog), and a hydrolase (AdaM) (Fig. 2A, 249
Table 2). To see if the genes (adaF-J) are needed for 2’-Cl PTN/2’-Amino dA 250
biosynthesis, the target region covering adaJ-M was deleted to give the mutant LG1 251
as confirmed by PCR (Fig. S2F-G), and the mutant (LG1) was then inoculated for 252
further analysis of the metabolites. As expected, the LC-MS results indicated that the 253
sample of the strain LG1 could not generate the characteristic peaks of 2’-Cl PTN, 254
PTN, and 2’-Amino dA, which are present in the sample of wild type strain (Fig. 2B). 255
All of the data demonstrate that the target gene cluster is simultaneously 256
responsible for the biosynthesis of 2’-Cl PTN, PTN, and 2’-Amino dA in Actinomadura 257
sp. ATCC 39365. 258
259
Engineered production of 2’-Cl PTN as well as 2’-Amino dA in a heterologous host 260
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To achieve engineered production of 2’-Cl PTN and 2’-Amino dA in a heterologous 261
host, a positive cosmid 3G12, housing the probable complete ada gene cluster, was 262
screened from the genomic library of Actinomudura sp. ATCC 39365 by a narrow-263
down PCR strategy (20), and introduced into the heterologous host S. 264
aureochromogenes CXR14 (21). Subsequently, the recombinant strain (CXR14::3G12), 265
confirmed by PCR, was fermented for further metabolic analysis, and the LC-MS 266
results indicated that the targeted [M+H]+ ions of 2’-Cl PTN (m/z=303.0825), PTN 267
(m/z=269.1220), and 2’-Amino dA (m/z=267.1175) could be clearly detected from 268
the sample of the CXR14::3G12 recombinant. In addition, tandem MS/MS analysis of 269
the target peaks indicated that their individual major fragment ions are fully 270
consistent with the fragmentation patterns of those antibiotics produced by 271
Actinomudura sp. ATCC 39365 (Fig. S3A-C). However, we could not detect the 272
[M+H]+ ions of 2’-Cl PTN, PTN, and 2’-Amino dA from the sample of the strain 273
without the target gene cluster (CXR14::pJTU2463b, negative control) (Fig. 3). All of 274
the data has demonstrated that the CXR14::3G12 recombinant is conferred with the 275
ability to produce the antibiotics 2’-Cl PTN, PTN, and 2’-Amino dA, and also 276
determined that the cosmid 3G12 contains the complete gene cluster essential for 277
the biosynthesis of 2’-Cl PTN and 2’-Amino dA. 278
279
The minimal 13-gene cluster is essential for 2’-Cl PTN and 2’-Amino dA biosynthesis 280
To further define the minimal gene cluster for the biosynthesis of 2’-Cl PTN and 2’-281
Amino dA, we determined the inserted foreign fragment of 3G12 cosmid via 282
terminal sequencing (Fig. 2A). On the basis of bioinformatic analysis, adaA-E 283
comprises a transcription unit, and orf-1 in 3G12 is incomplete, suggesting that this 284
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gene is the left boundary of the ada gene cluster (Fig. 2A). We then investigated the 285
downstream component of the ada gene cluster, adaLM compose a transcriptional 286
unit, implicating that orf+1 is likely unrelated with 2’-Cl PTN and 2’-Amino dA 287
biosynthesis. To validate the assumption, we then mutated this gene directly on 288
3G12 cosmid and introduced the resultant 3G12 derivative into S. 289
aureochromogenes CXR14 to see if it is necessary for 2’-Cl PTN and 2’-Amino dA 290
biosynthesis. The LC-MS results showed that the sample of the mutant 291
(CXR14::3G12/Δorf1) is capable of generating the apparent 2’-Cl PTN, PTN, and 2’-292
Amino dA [M+H]+ ions, confirming the unrelated role of this gene with the 293
biosynthesis of the target antibiotics (Fig. 3). These combined data unambiguously 294
established that the 13 genes (adaA-M) spanning a ca. 14.4-kb region constitute the 295
minimal ada gene cluster for 2’-Cl PTN and 2’-Amino dA biosynthesis (Fig. 2A, Fig. 3). 296
297
Mutational analysis of the ada gene cluster reveals 2’-Cl PTN and 2’-Amino dA 298
utilize independent biosynthetic pathways 299
For systematic insight into the genetic roles of the ada genes during 2’-Cl PTN and 300
2’-Amino dA biosynthesis, we directly conducted in-frame deletion of all the target 301
individual genes on 3G12 via PCR-targeting technology (18), and the resulting 3G12 302
derivatives, verified by PCR, were independently conjugated into S. 303
aureochromogenes CXR14 (Fig. S4). After fermentation for 6 d, the samples of the 304
related recombinants were subjected to LC-MS analysis. The results indicated that 305
mutation of adaA, adaB, adaC, or adaL completely abolishes 2’-Cl PTN, PTN, and 2’-306
Amino dA production (Fig. 3), demonstrating the essential functional roles of them 307
for the three antibiotics’ biosynthesis, and mutation of the structural genes adaF, 308
14
adaG, or adaJ results in the nonproduction of 2’-Amino dA, but interestingly, the 309
production of 2’-Cl PTN and PTN remains almost unaffected, suggesting that they are 310
specially required for 2’-Amino dA biosynthesis. Moreover, we revealed that 311
individual mutation of the transporter genes including adaD, adaH, or adaI prevents 312
2’-Amino dA production (Fig. 2A, Fig. 3), implying that the gene products act as 313
specific transporters of 2’-Amino dA. 314
Furthermore, we found the production of 2’-Amino dA and 2’-Cl PTN remains 315
almost intact for the ΔadaK or ΔadaM mutant (Fig. 3), suggesting that there might 316
be other genes that encode alternative functional products capable of performing 317
the same roles as adaK or adaM. Curiously, as for ΔadaE mutant, LC-MS analysis 318
reveals that the production of 2’-Cl PTN and 2’-Amino dA is completely abrogated, 319
while that of PTN is barely unaffected, implicating that this AdaE enzyme might play 320
potential role in the chlorination of PTN to produce 2’-Cl PTN, however, the precise 321
mechanism on how this reaction occurs has remained enigmatic. All together, our 322
genetic data suggest that 2’-Amino dA and 2’-Cl PTN arise from independent 323
biosynthetic pathways, and 2’-Amino dA biosynthesis is strictly dependent on the 324
production of 2’-Cl PTN (Fig. 3). 325
326
In silico analysis of the PTN and Ara-A biosynthetic gene cluster 327
Sequence analysis indicates that the G+C content of the minimal ada gene cluster 328
is relatively high (73.64%) but similar to that of the typical genome of Actinomycetes. 329
In ada gene cluster, adaA-E, adaF-J, and adaLM constitute individual transcriptional 330
units according to bioinformatic analysis, whereas adaK is a standalone gene which 331
is located in the middle of the ada gene cluster (Fig. 2A). 332
15
As suggested by genetic investigation and in silico analysis, adaABCEKL are 333
proposed to be involved in 2’-Cl PTN biosynthesis. Of their products, AdaA, AdaB, 334
and AdaC correspond to the individual enzymes, PenC (saicar synthetase, 72% 335
identity), PenB (short-chain dehydrogenase, 70% identity), and PenA (ATP 336
phosphoribosyltransferase, 58% identity), in PTN biosynthetic pathway from S. 337
antibioticus NRRL 3238 (Fig. 2A, Table 2). Another enzyme AdaL also indicates 338
significant homology to PenA with 53% identity, and it is deduced to be needed for 339
2’-Cl PTN biosynthesis. As for adaK, it encodes a protein showing 75% identity to 340
HMPREF1486_01538 annotated as phosphoribosyl isomerase (HisA) from 341
Streptomyces sp. WM4235 (Fig. 2A, Table 2). Very attractively, AdaE exhibits 54% 342
identity to ADL15_15700, an annotated cation/H+ antiporter, from S. venezuelae 343
ATCC 15439 (Fig. 2A, Table 2), but how does this protein participate in 2’-Cl PTN 344
biosynthesis is currently unknown. 345
Four other structural genes adaFGJM are likely to be required for 2’-Amino dA 346
biosynthesis (Fig. 2A, Table 2). AdaF codes for a protein showing 53% identity to an 347
aminotransferase, COCOR_00673 from Corallococcus coralloides DSM 2259, and this 348
enzyme is directly responsible for the transfer of amino group during 2’-Amino dA 349
biosynthesis. AdaG has moderate homology (28% identity) to PCL1391_5850 (a 350
predicted dehydrogenase) of Pseudomonas chlororaphis, and AdaJ shows significant 351
homology (54% identity) to Caci_7624, a putative NUDIX hydrolase from 352
Catenulispora acidiphila. AdaJ may trigger the biosynthesis of 2’-Amino dA by 353
hydrolyzing ATP to form AMP. In ada gene cluster, three genes adaDHI encodes 354
putative transporters, which are particularly responsible for the transportation of 355
the product 2’-Amino dA. 356
16
Biochemical characterization of AdaJ as an ATP NUDIX hydrolase 357
In silico analysis indicated that AdaJ contains a highly conserved 23-residue NUDIX 358
motif (GX5EX7REUXEEXGW) (Fig. S5), which functions as a metal binding and catalytic 359
site, therefore, this enzyme presumably function as a NUDIX hydrolase. To see if 360
AdaJ executes such functional role, it was overexpressed in E. coli with a fusion MBP 361
(Maltose binding protein) tag, and purified to near-homogeneity (Fig. 4A). As 2’-362
Amino dA is a adenosine analog, implicating that ATP is most likely the substrate of 363
AdaJ, moreover, NUDIX hydrolases usually require a divalent cation, such as Mg2+ or 364
Mn2+, for their activity, we therefore test its activity in vitro using ATP as substrate 365
and Mg2+ as divalent cation, as expected, the results showed that AdaJ is capable of 366
converting ATP to form a new peak at RT=21.0 min, which is consistent with that of 367
AMP authentic standard (RT=21.3 min) (Fig. S6A). Further LC-MS analysis exhibited 368
that the new peak could produce a characteristic [M+H]+ ion at m/z=348.0704, with 369
fragment at 135.9938, which fully agree with those of the AMP authentic standard 370
(Fig. S6B-C). However, the negative control could not generate the characteristic 371
AMP peak. 372
Next, we evaluated the influence of divalent cation on AdaJ activity. Of all divalent 373
cations selected, including Co2+, Mg2+, Mn2+, Fe2+, Cu2+, and Zn2+, and surprisingly, we 374
found that Co2+ is capable of maintaining the maximal activity for AdaJ, thereof 375
suggesting that AdaJ is a non-canonical NUDIX hydrolase using Co2+ as the most 376
preferred metallic factor (Fig. 4B-C). We subsequently tested the substrate flexibility 377
of the enzyme, and we found that AdaJ could also consume dATP and GTP as the 378
substrate, but it could not recognize ADP as a substrate (Fig. 4D). Taken together, 379
17
our biochemical data verified that AdaJ functions as a distinctive NUDIX hydrolase 380
that initiates the biosynthesis of 2’-Amino dA by hydrolysis of ATP to AMP. 381
382
The host adenosine deaminase ADA1 contributes to 2’-Amino dA deamination and 383
is relatively insensitive to PTN 384
We found that Actinomadura sp. ATCC 39365 is capable of producing 2’-Amino dI 385
in a lower yield (only detectable by LC-MS), despite the fact that 2’-Cl PTN and PTN 386
are both powerful adenosine deaminase inhibitors. In addition, why the adaE 387
mutant abolishes the capability of producing 2’-Amino dA? To address which 388
adenosine deaminase of the host contributes to the phenotypes, we thus select a 389
versatile Ara-A/adenosine deaminase SanADA3 (Accession no: KT591401) from S. 390
antibioticus (19) as probe to conduct BlastP analysis against the genome of 391
Actinomadura sp. ATCC 39365, which leads to the identification of four homologs. Of 392
them, ADA1 exhibits the highest homology (62% identity) to SanADA3 (Fig. S7A), 393
implicating that this enzyme is most likely to govern the deamination of 2’-Amino dA. 394
To test the assumption, it was overexpressed in E. coli and purified to near 395
homogeneity (Fig. 5A), and we then test its activity in vitro. As anticipated, the 396
results established that ADA1 converts adenosine/2’-Amino dA to inosine/2’-Amimo 397
dI, confirming its functional role as an adenosine/2’-Amino dA deaminase (Fig. 5B-D, 398
Fig. S7B-C). To further see if PTN is capable of protecting 2’-Amino dA from 399
deamination by ADA1, the reactions containing PTN (final conc. 0.01 mM, 0.1 mM, or 400
1 mM) and the substrates (Adenosine or 2’-Amino dA) were initiated by adding ADA1. 401
HPLC results indicated that PTN is not able to protect adenosine from deamination at 402
all even in the presence of 1 mM PTN (Fig. 5C, Fig. S7D), while 2’-Amino dA can be 403
18
partially protected from deamination in the presence of PTN (0.1 mM, or 1 mM) (Fig. 404
5D, Fig. S7D). These data suggest that ADA1 functions as an adenosine/2’-Amino dA 405
deaminase, and is relatively insensitive to inhibition by PTN. 406
407
DISCUSSION 408
Earlier metabolic feeding experiments has established that 2’-Cl PTN and PTN 409
biosynthesis harbors a ring-expansion with an additional one-carbon unit (C-7) 410
insertion between C-6 and N-1 of the purine scaffold, and the one-carbon unit has 411
been previously demonstrated to be derived from C-1 of ribose (7, 22). More than 412
that, the 2’-PTN/PTN pathway was deduced to be closely related to that of the 413
primary L-histidine (23). The results of this study are consistent with previous work 414
for PTN pathway in S. antibioticus NRRL 3238 (19). Five enzymes including AdaA 415
(saicar synthetase), AdaB (short-chain dehydrogenase) AdaC (ATP 416
phophoribosyltransferase), AdaK (phophoribosyl isomerase) and AdaL (ATP 417
phophoribosyltransferase) are assigned to be involved in PTN pathway (Fig. 6A). The 418
PTN biosynthesis would start with condensation of dATP and PRPP (phosphoribiosyl 419
pyrophosphate) to from 1 by two enzymes AdaC and AdaL, and we propose that both 420
of them would collaborate together to fulfill the catalytic function accounting for the 421
fact that mutation of each one, either AdaC or AdaL, leads to the abolishment of PTN 422
production. Subsequently, compound 1 will be converted to 2 through sequential 423
reactions by three enzymes HisI (phosphoribosyl-AMP cyclohydrolase), HisE 424
(phosphoribosyl-ATP pyrophosphatase), and HisA (AdaK homolog, phosphosribosyl 425
isomerase) from histidine pathway. Interestingly, we find that the 2’-Cl PTN/PTN 426
pathway contains a HisA homolog (AdaK) as well, which is likely to perform identical 427
19
function role to HisA as confirmed by our genetic investigation. Compound 2 will be 428
catalyzed to 5 via the deduced intermediates 3 and 4 by unique reactions that is 429
proposed to be sequentially catalyzed by AdaA (saicar synthetase), which was 430
previously uncharacterized in nucleoside antibiotics biosynthesis (Fig. 6A). After 431
dephosphorylation by AdaM or an unknown enzyme, compound 6 is dehydrogenated 432
by AdaB (PenB homolog) to produce the target product PTN (Fig. 6A) (19). 433
2’-Cl PTN was documented as one of the few naturally-occurring nucleoside that 434
contained a chloro group (24), but the biosynthetic origin and molecular mechanism 435
of the chlorination has remained obscure for decades (24). Previous metabolic 436
labeling studies has established that the inorganic chloride could be directly utilized 437
during 2’-Cl PTN biosynthesis (4). Furthermore, it was proposed that several 438
alternative chlorination mechanisms would be potentially employed for such 439
chlorination, including (i) stereo-selective insertion of chloro group involves a 440
choloro-peroxidase-catalyzed reaction, (ii) conversion of 2’-Amino dA to 2’-441
diazoadenosine whose diazonium group can be displaced by a nucleophilic attack by 442
a chloride ion, (iii) involving a cation radical enzyme-catalyzed reaction(4). However, 443
based on the in silico analysis, we cannot identify an obviously classical halogenase in 444
the 2’-Cl PTN pathway, which has reversely conferred such chlorination with more 445
mysteries. Incredibly, our genetic studies indicated that adaE (encoding cation/H+ 446
antiporter) is likely related with the chlorination, which is further supported by the 447
identification of the candidate homologous enzymes in the pathways of ascamycin 448
(AcmU), and nucleocidin (NucU), we thus speculate that AdaE should be related with 449
tailoring chlorination step (Fig. 6A), and there might be a potential haloperoxidase 450
enzyme existed in the both original and heterologous expression host, which assists 451
20
in the chlorination, and related research is now in intensive progress in our 452
laboratory. 453
Previous metabolic labeling experiments has demonstrated that adenosine is the 454
direct precursor for the biosynthesis of 2’-Amino dA, and adenosine is first 455
dehydrogenated to form a putative 2’-keto adenosine, which is then transaminated 456
to generate the end product 2’-Amino dA (4). In the present study, this is essentially 457
correct. Four enzymes including AdaJ (NUDIX hydrolase), AdaM (hydrolase), AdaG 458
(dehydrogenase), and AdaF (aminotransferase) are defined to participate in the 459
biosynthesis of 2’-Amino dA. We propose that the biosynthesis of 2’-Amino dA is 460
initiated by AdaJ, which catalyzes the hydrolysis of ATP to AMP with release of a 461
pyrophosphate, and the catalytic reaction has been well characterized in our present 462
study. The intermediate AMP is then dephosphorylated to adenosine by AdaM or 463
alternative enzyme(s), which undergoes the following dehydrogenation to form the 464
2’-keto adenosine intermediate (even though it could not be detectable due to its 465
poor production or other unknown reasons). Finally, 2’-keto adenosine is catalyzed 466
by a transamination step to accomplish the biosynthesis of the end nucleoside 2’-467
Amino dA (Fig. 6B). 468
The attractive phenomenon of the concomitant production of the purine 469
nucleoside pairs, as exemplified by PTN-AraA, coformycin-formycin, and 2’-Cl PTN-2’-470
Amino dA, is more widely distributed than we imagined (19). Utilization of the three 471
conserved enzymes (AdaA, AdaB, and AdaC) as valuable probes could lead to the 472
discovery of additional pathways of the potential PTN-related antibiotics pairs from 473
the reservoir of sequenced microbial genomes. Notably, the advent of rapid and 474
affordable DNA-sequencing will certainly accelerate the traditional process for the 475
21
discovery of new drugs related with PTN-related antibiotic pairs. Moreover, the 476
enzymatic reactions of halogenation reaction in the biosynthesis of 2’-Cl PTN, 477
ascamycin, and nucleocidin has remained obscure for a half century (25, 26), and our 478
identifying a probable cation/H+ antiporter AdaE that plays a potential role in the 479
chlorination in 2’-Cl PTN will not only open a door for further illumination of the 480
entirely unknown mechanism for halogenation chemistry, but may also used as 481
enzyme probe for the discovery of more halogenated natural products. 482
It is interesting that why the adaM mutant also retains the ability to produce 2’-483
Amino dA, and we conclude that the functional role of AdaM (AMP hydrolase) might 484
be replaced by other alternative homologs. We also report a seemingly paradoxical 485
finding, namely that the adaE mutant lacks the ability to synthesize 2’-Amino dA, 486
since this gene is merely proposed to be essential for 2’-Amino dA? We tentatively 487
propose that once the tailoring chlorination step terminates during 2’-Cl PTN 488
biosynthesis, the metabolic flux of cell factory can only turn to PTN biosynthesis, 489
however, the host adenosine deaminase is not sensitive to this nucleoside analog, as 490
a result, adenosine, once synthesized, will be immediately deaminated to inosine for 491
purine recycling. 492
In summary, we report the finding and functional analysis of a 13-gene cluster 493
essential for 2’-Cl PTN and 2’-Amino dA biosynthesis. We further determine that 494
these two nucleosides arise from independent biosynthetic pathways, and provide 495
biochemical proof that the adenosine deaminase ADA1 is capable of catalyzing the 496
deamination of 2’-Amino dA, but this enzyme is not highly sensitive to the inhibition 497
of PTN. We have also illustrated that AdaJ (NUDIX hydrolase) governs the initial step 498
in 2’-Amino dA biosynthesis. We anticipate that uncovering the precise logic 499
22
underlying the biosynthesis of 2’-Cl PTN and 2’-Amino dA will be of great potential 500
for the combinatorial biosynthesis of this group of nucleoside antibiotics pairs with 501
modified activity and selectivity. 502
503
ACKNOWLEDGMENTS 504
This work was supported by grants the National Science Foundation of China 505
(31270100, 21402146), Hubei Provincial Natural Science Foundation of China 506
(2016CFB458), and Wuhan Youth Chenguang Program of Science and Technology 507
(2015070404010181). 508
509
REFERENCES 510
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3. Winn M, Goss RJ, Kimura K, Bugg TD. 2010. Antimicrobial nucleoside antibiotics targeting 516 cell wall assembly: recent advances in structure-function studies and nucleoside 517 biosynthesis. Nat Prod Rep 27:279-304. 518
4. Suhadolnik RJ, Pornbanlualap S, Baker DC, Tiwari KN, Hebbler AK. 1989. Stereospecific 519 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-520 amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin. Arch Biochem Biophys 521 270:374-82. 522
5. Matsuyama K, Takahashi Y, Yamashita M, Hirano A, Omura S. 1979. 2'-Amino-2'-523 deoxyadenosine produced by a strain of Actinomadura. J Antibiot (Tokyo) 32:1367-9. 524
6. Omura S, Imamura N, Kuga H, Ishikawa H, Yamazaki Y, Okano K, Kimura K, Takahashi Y, 525 Tanaka H. 1985. Adechlorin, a new adenosine deaminase inhibitor containing chlorine 526 production, isolation and properties. J Antibiot (Tokyo) 38:1008-15. 527
7. Hanvey JC, Hardman JK, Suhadolnik RJ, Baker DC. 1984. Evidence for the conversion of 528 adenosine to 2'-deoxycoformycin by Streptomyces antibioticus. Biochemistry 23:904-7. 529
23
8. Nakamura H, Koyama G, Iitaka Y, Ono M, Yagiawa N. 1974. Structure of coformycin, an 530 unusual nucleoside of microbial origin. J Am Chem Soc 96:4327-8. 531
9. Tunac JB, Underhill M. 1985. 2'-Chloropentostatin: discovery, fermentation and biological 532 activity. J Antibiot (Tokyo) 38:1344-9. 533
10. Utagawa T, Morisawa H, Yamanaka S, Yamazaki A, Hirose Y. 1985. Enzymatic-Synthesis of 534 Nucleoside Antibiotics .4. Microbial Synthesis of Purine 2'-Amino-2'-Deoxyribosides. 535 Agricultural and Biological Chemistry 49:2711-2717. 536
11. Utagawa T, Morisawa H, Yamanaka S, Yamazaki A, Yoshinaga F, Hirose Y. 1985. Enzymatic-537 Synthesis of Nucleoside Antibiotics .2. Microbial Synthesis of Purine Arabinosides and 538 Their Biological-Activity. Agricultural and Biological Chemistry 49:2167-2171. 539
12. Nakanishi T, Tomita F, Furuya A. 1977. Uptake of 2'-amino-2'-deoxyguanosine by 540 Escherichia coli and its competition by guanosine. J Antibiot (Tokyo) 30:736-42. 541
13. Pugh LH, Gerber NN. 1963. The effect of 3'-amino-3'-deoxyadenosine against ascitic 542 tumors of mice. Cancer Res 23:640-7. 543
14. Wu P, Wan D, Xu G, Wang G, Ma H, Wang T, Gao Y, Qi J, Chen X, Zhu J, Li YQ, Deng Z, Chen 544 W. 2017. An Unusual Protector-Protege Strategy for the Biosynthesis of Purine 545 Nucleoside Antibiotics. Cell Chem Biol 24:171-181. 546
15. Schaumberg JP, Hokanson, G. C., French, J. C. 1985. 2'-Chloropentostatin, a New Inhibitor 547 of Adenosine Deaminase. J Org Chem 50:6. 548
16. Green MR, Sambrook J. 2002. Molecular Cloning: a Laboratory Manual:3rd ed., Cold 549 Spring Harbor Laboratory Press, NY. 550
17. Kieser T, Bibb MJ, Chater KF, Butter MJ, Hopwood. DA. 2000. Practical Streptomyces 551 Genetics:2nd ed., John Innes Foundation, Norwich, United Kingdom. 552
18. Gust B, Challis GL, Fowler K, Kieser T, Chater KF. 2003. PCR-targeted Streptomyces gene 553 replacement identifies a protein domain needed for biosynthesis of the sesquiterpene 554 soil odor geosmin. Proc Natl Acad Sci U S A 100:1541-6. 555
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20. Cheng L, Chen W, Zhai L, Xu D, Huang T, Lin S, Zhou X, Deng Z. 2011. Identification of the 559 gene cluster involved in muraymycin biosynthesis from Streptomyces sp. NRRL 30471. 560 Mol Biosyst 7:920-7. 561
21. Qi J, Wan D, Ma H, Liu Y, Gong R, Qu X, Sun Y, Deng Z, Chen W. 2016. Deciphering 562 Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated 563 with Tandem Reduction and Sequential Hydroxylation. Cell Chem Biol 23:935-44. 564
24
22. Hanvey JC, Hawkins ES, Tunac JB, Dechter JJ, Baker DC, Suhadolnik RJ. 1987. Biosynthesis 565 of 2'-deoxycoformycin: evidence for ring expansion of the adenine moiety of adenosine 566 to a tetrahydroimidazo[4,5-d][1,3]diazepine system. Biochemistry 26:5636-41. 567
23. Hanvey JC, Hawkins ES, Baker DC, Suhadolnik RJ. 1988. 8-Ketodeoxycoformycin and 8-568 ketocoformycin as intermediates in the biosynthesis of 2'-deoxycoformycin and 569 coformycin. Biochemistry 27:5790-5. 570
24. van Pee KH. 1996. Biosynthesis of halogenated metabolites by bacteria. Annu Rev 571 Microbiol 50:375-99. 572
25. Zhao C, Qi J, Tao W, He L, Xu W, Chan J, Deng Z. 2014. Characterization of biosynthetic 573 genes of ascamycin/dealanylascamycin featuring a 5'-O-sulfonamide moiety in 574 Streptomyces sp. JCM9888. PLoS One 9:e114722. 575
26. Zhu XM, Hackl S, Thaker MN, Kalan L, Weber C, Urgast DS, Krupp EM, Brewer A, Vanner S, 576 Szawiola A, Yim G, Feldmann J, Bechthold A, Wright GD, Zechel DL. 2015. Biosynthesis of 577 the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the 578 bldA-Specified Leu-tRNA(UUA) Molecule. Chembiochem 16:2498-506. 579
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583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600
25
Table 1. Strains, plasmids and cosmids used in this study. 601
Strain/ Plasmid / Cosmid
Relevant characteristics* Reference or source
Strain Actinomadua sp.
ATCC 39365 LG1 S. aureochromogenes CXR14
Wild-type strainThe adaJ, adaK, adaL, adaM genes deleted in ATCC 39365 An industrial polyoxin producer with the entire polyoxin gene cluster deleted
(4) This study (21)
CXR14::3G12 CXR14 strain containing 3G12 This studyCXR14::3G12lΔadaE CXR14 strain containing 3G12lΔadaE This studyCXR14::3G12lΔadaD CXR14 strain containing 3G12lΔadaD This studyCXR14::3G12lΔadaC CXR14 strain containing 3G12lΔadaC This studyCXR14::3G12lΔadaB CXR14 strain containing 3G12lΔadaB This studyCXR14::3G12lΔadaA CXR14 strain containing 3G12lΔadaA This studyCXR14::3G12lΔadaF CXR14 strain containing 3G12lΔadaF This studyCXR14::3G12lΔadaG CXR14 strain containing 3G12lΔadaG This studyCXR14::3G12lΔadaH CXR14 strain containing 3G12lΔadaH This studyCXR14::3G12lΔadaI CXR14 strain containing 3G12lΔadaI This studyCXR14::3G12lΔadaJ CXR14 strain containing 3G12lΔadaJ This studyCXR14::3G12lΔadaK CXR14 strain containing 3G12lΔadaK This studyCXR14::3G12lΔadaL CXR14 strain containing 3G12lΔadaL This studyCXR14::3G12lΔadaM CXR14 strain containing 3G12lΔadaM This studyCXR14::3G12lΔorf+1 CXR14 strain containing 3G12lΔorf+1 This studyE. coli DH10B Cloning host Gibco-BRL
BW25113/pIJ790 λRED(gam,beta,exo),cat,araC,rep101 (18) BL21(DE3)/pLysE F-, ompT, hsdSB(rB
-mB-), gal, dcm(DE3), pLysE (CmlR) STRATAGENE
Rosetta(DE3)/pLysS F-, ompT, hsdSB(rB-mB
-), gal, dcm λ(DE3 [lacI lacUV5-T7 gene1 ind1 sam7 nin5]) pLysS
Novagen
ET12567 (pUZ8002) dam, dcm, hsdM, hsdS, hsdR, cat, tet, neo; helper strain for intergeneric conjugation
(17)
EPI300-T1R Cosmid library host cell EpicenterPlasmids pEASY-Blunt pUCori, lacZ, f1 ori, neo, bla TransGen
Biotech pJTU2463b int, aac(3)IV, oriT, RK2, phiC31, attP (20) pOJ446 aac(3)IV, SCP2, reppMB1*, attФC31, ori T (27) pET28a neo, rep pMB1, T7 promoter Novagen pSJ8 lac, MBP, f1 ori, bla
26
3G12 Cosmid containing the entire ada gene cluster This studypLG001 pOJ446 derivative with insertion of 1.9-kb XbaI-BglII
engineered PCR fragment of left arm This study
pLG002 pLG001 derivative carrying a BglII engineered PCR fragment containing 2.5-kb of right arm
This study
pLG003 pLG002 derivative with insertion of a BglII fragment containing neo
This study
pET28a/ADA1 pET28a derivative carrying a NdeI-EcoRI fragment containing ada1 encoding 357 aa
This study
pSJ8/adaJ pSJ8 derivative carrying a EcoRI-HindIII fragment containing adaJ encoding 161 aa
This study
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
27
Table 2. Deduced functions of the open reading frames in the ada gene cluster 620
Protein aa Proposed function Homolog, Origin
Identity,
similarity
(%)
Accession no.
Orf-1 1061 Hypothetical protein BN2537_3105, Streptomyces
venezuelae ATCC 15439 34,46
CUM37070
AdaE 479 Cation/H+ antiporter ADL15_15700, Actinoplanesa
wajinensis NRRL B-16712 54,67
KUL34520
AdaD 402 MFS transporter SAMN05421874_102571,Nonom
uraeamaritima 72,82
SDJ65310
AdaC 295 ATP phosphoribosyl-
transferase
PenA, Streptomyces antibioticus
NRRL 3238 58,71 AKA87340
AdaB 234 Short-chain dehydrogenase PenB, Streptomyces antibioticus
NRRL 3238 70,82 AKA87339
AdaA 239 SAICAR synthetase PenC, Streptomyces antibioticus
NRRL 3238 72,81 AKA87338
AdaF 425 Aminotransferas COCOR_00673, Corallococcus
coralloides DSM 2259 53,70 AFE09038
AdaG 351 Dehydrogenase PCL1391_5850, Pseudomonas
chlororaphis subsp. piscium 28,46 KZO46392
AdaH 595 ABC transporter, partial SAMN05421811_12174,
Nonomuraea wenchangensis 46,63 SEU43234
AdaI 592 ABC transporter Trad_2349,Truepera radiovictrix
DSM 17093 49,68 ADI15458
AdaJ 161 NUDIX hydrolase Caci_7624, Catenulispora
acidiphila DSM 44928 54,66 ACU76448
AdaK 257 Phosphoribosyl isomerase
A
HMPREF1486_01538,
Streptomyces sp. HPH0547 75,85
EPD96008
AdaL 288 ATP phosphoribosyl-
transferase
ADK55_17455, Streptomyces sp.
WM4235 54,68 KOU52239
AdaM 264 Hydrolase ADK55_17445, Streptomyces sp.
WM4235 62,71
KOU52238
Orf1 339 ABC transporter substrate-
binding protein
BCD48_37375, Frankia sp.
BMG5.36 55,68 OHV64536
621
622
623
624
28
Figures and Legends 625
626
Figure 1. Chemical structures of relevant antibiotics. 627
(A) Chemical structures of related antibiotic pairs. The upper and lower structures 628
constitute antibiotic pairs which are co-produced by particular strain. 2’-Cl 629
pentostatin and 2’-amino-2’-deoxyadenosine (2’-Amino dA) pair are concomitantly 630
produced by Actinomudura sp. ATCC 39365; PTN and Ara-A pair are produced by S. 631
antibioticus NRRL 3238; Coformycin and formycin pair are produced by S. 632
kaniharenes ATCC 21070 or by Nocardia interforma ATCC 21072. (B) Chemical 633
Structure of 2’-Amino dI, 3’-Amino dA, and 2’-Amino dG. 2’-Amino dI, 2’-amino-2’-634
deoxyinosine; 2’-Amino dG, 2’-amino-2’-deoxyguanosine; 3’-Amino dA, 3’-amino-3’-635
deoxyinosine. 636
29
637
Figure 2. Genetic organization and validation of the 2’-Cl PTN and 2’-Amino dA 638
biosynthetic gene cluster (ada). 639
(A) Genetic organization of the ada gene cluster. The insertion region of the positive 640
cosmid 3G12 was indicated on the top of the ada gene cluster, and the conserved 641
genes in the shade region are used for the identification of the ada gene cluster. (B) 642
LC-MS analysis of the target metabolites produced by Actinomudura sp. LG1 mutant. 643
(C) MS analysis of the 2’-Cl PTN ion generated by the sample of Actinomudura sp. 644
ATCC 39365. 645
646
647
648
649
650
30
651
Figure 3. Genetic investigation of the 2’-Cl PTN and 2’-Amino dA biosynthetic 652
pathways. 653
Extract ion chromatography (EIC) analysis of the metabolites produced by S. 654
aureochromogenes CXR14::3G12 and its variants. ΔadaE refers to the sample from 655
the strain of S. areochromogenes CXR14 containing 3G12/ΔadaE, in which adaE was 656
in-frame deleted via PCR-targeting strategy, likewise, other related samples are 657
correspondingly assigned; WT, wild type strain of ATCC 39365; 3G12, the strain of S. 658
areochromogenes CXR14 containing cosmid 3G12; 2463b means the strain of S. 659
areochromogenes CXR14 containing pJTU2463b as negative control. 660
661
31
662
Figure 4. Biochemical characterization of AdaJ as an ATP NUDIX hydrolase. 663
(A) SDS-PAGE analysis of the purified AdaJ. (B) Relative activity of AdaJ with ATP as 664
substrate and different divalent cations as metallic cofactor. (C) HPLC traces of AdaJ 665
catalyzed reaction with ATP as substrate and Co2+ as cofactor. (i) AdaJ catalyzed 666
reaction with ATP as substrate and Co2+ as cofactor; (ii) the negative control without 667
enzyme added; (iii) the authentic standard of AMP; (iv) the authentic standard of ATP. 668
(D) Evaluation of the AdaJ activity against different substrates; Co2+ was utilized as 669
the metallic cofactor. The error bars represent the SD from at least three different 670
experiments. 671
672
673
32
674
Figure 5. In vitro characterization of ADA1 as the adenosine/2’animo-dA deaminase 675
which is not much sensitive to PTN. 676
(A) SDS-PAGE analysis of Actinomadura sp. ATCC 39365 adenosine deaminase 1 677
(abbreviated as ADA1). (B) Schematic of ADA1-catalyzed reaction. (C) HPLC traces of 678
ADA1-catalyzed reaction with adenosine as substrate. PTN is not an effective ADA1 679
inhibitor. (i), the authentic standard of inosine; (ii), ADA1 reaction with 1 mM PTN 680
(final conc.) added; (iii), ADA1 reaction with 0.1 mM PTN (final conc.) added; (iv), 681
ADA1 reaction with 0. 01 mM PTN (final conc.) added; (v), ADA1 reaction without 682
PTN added; (vi), negative control without enzyme and PTN added; (vii), negative 683
control without enzyme but with 1 mM PTN added. (D) HPLC traces of ADA1-684
catalyzed reaction with 2’-Amino dA as substrate and PTN as inhibitor. (i), the 685
authentic standard of 2’-Amino dI; (ii), ADA1 reaction with 1 mM PTN (final conc.) 686
added; (iii), ADA1 reaction with 0.1 mM PTN (final conc.) added; (iv), ADA1 reaction 687
33
with 0. 01 mM PTN (final conc.) added; (v), ADA1 reaction without PTN added; (vi), 688
negative control without enzyme and PTN added; (vii), negative control without 689
enzyme but with 1 mM PTN added. 690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
34
710
Figure 6. Proposed biosynthetic pathways to 2’-Cl PTN (A) and 2’-Amino dA (B). 711
In the proposed 2’-Cl PTN pathway, the initial step is consistent with the previous 712
metabolite labeling experiments, and the following steps are proposed according to 713
in silico analysis. 714
715
716
717