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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: wqchen@whu.edu.cn, 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

54

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

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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

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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

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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

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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

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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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2. Chen W, Qi J, Wu P, Wan D, Liu J, Feng X, Deng Z. 2016. Natural and engineered 513 biosynthesis of nucleoside antibiotics in Actinomycetes. J Ind Microbiol Biotechnol 514 43:401-17. 515

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

19. Wu P, Wan D, Xu G, Wang G, Ma H, Wang T, Gao Y, Qi J, Chen X, Zhu J, Li Y-Q, Deng Z, 556 Chen W. 2017. An unusual protector-protege strategy for the biosynthesis of purine 557 nucleoside antibiotics. Cell Chem Biol 24. 558

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

27. Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, Schoner BE. 1992. Plasmid cloning 580 vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 581 116:43-9. 582

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