Biosynthesis of 2′-Chloropentostatin and 2′-Amino-2′-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365

Abstract

2=-Chloropentostatin (2=-Cl PTN, 2=-chloro-2=-deoxycoformycin) and 2=-amino-2=-deoxyadenosine (2=-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2=-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2= of ribose, and 2=-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2=-Cl PTN and 2=-amino dA. Further systematic genetic investigations suggest that 2=-Cl PTN and 2=-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H ϩ antiporter, AdaE, is involved in the chlorination step during 2=-Cl PTN biosynthesis. Notably, we demonstrate that 2=-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2=-amino dA to inosine/2=-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2=-Cl PTN and 2=-amino dA production.IMPORTANCE 2=-Cl PTN/PTN and 2=-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.KEYWORDS 2=-chloropentostatin, 2=-amino-2=-deoxyadenosine, nucleoside antibiotics, biosynthesis, Nudix hydrolase, adenosine deaminase N ucleoside antibiotics are a large family of important microbial natural products harboring wide-range biological properties and distinctive structural features (1-3). Their biosynthesis generally follows a succinct logic by sequential enzymatic modification of nucleosides or nucleotides originating from primary metabolisms (1). Usually, nucleosides and nucleotides play pleiotropic roles in most fundamental cellular metabolisms; therefore, nucleoside antibiotics are able to target the biosynthesis of diverse biomacromolecules, including nucleic acids, proteins, and glycans (1).

An Fe2+‐ and α‐Ketoglutarate‐Dependent Halogenase Acts on Nucleotide Substrates

“…It is believed that 8 and 10 may share the same precursor, possibly from the primary metabolite pathway. The semi‐purified fractions containing 8 , 9 , and 10 were subjected to the analysis of high‐resolution mass spectrometry (HRMS) with the mass‐to‐charge ratio ( m / z ) of 303.0837, 269.1244, and 267.1168, respectively, fully consistent with the previous results …”

“…Previous work by Gao et al . revealed that the adechlorin biosynthetic gene cluster ( ade BGC) was composed of 13 genes ( ade A‐ ade M), but none of these were identified to encode the required chlorinase (Figure A).…”

Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

“…FOR-A and COF were formerly reported to be co-produced by the actinobacteria, Nocardia interforma ATCC 21072 and S. kaniharaensis ATCC 21070 (7, 10), implicating that the genes for their biosyntheses, as those of other purine nucleoside antibiotic pairs, should also constitute a gene cluster (11, 12), Additionally, COF and PTN, featuring significant structural resemblance, were recently revealed to share highly-similar biosynthetic pathways (20).…”

“…This intriguing co-biosynthetic phenomenon was also documented for other purine nucleoside antibiotics pairs, including pentostatin (PTN) and arabinofuranosyl adenine (Ara-A) (produced by Streptomyces antibioticus NRRL 3238) (11), 2’-Cl PTN and 2’-amino-2’-deoxyadenosine (produced by Actinomadura sp. ATCC 39365) (12), and fungus-produced PTN and cordycepin (13), which all employ an unusual but general protector-protégé strategy, i.e. PTN may protect Ara-A (taking this antibiotic-pair as example) from deamination by the housekeeping adenosine deaminase (11–13).…”

Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes

“…strain ATCC 39365) ( Fig. 1A) (11), as well as by the fungus-derived PTN and cordycepin pair (produced by Cordyceps militaris and other fungal strains) (12). In addition, we have previously shown that a short-chain dehydrogenase and a SAICAR (phosphoribosylaminoimidazolesuccinocarboxamide) synthetase are highly conserved for the biosynthesis of the heterocyclic 1,3-diazepine ring of the PTN-related compounds (8).…”