Abstract
BackgroundTubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, highlights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond. However, the molecular logic underlying the biosynthesis of this antibiotic has remained poorly understood.ResultsHere, we report the discovery and characterization of the TBN biosynthetic pathway from Streptomyces tubercidicus NBRC 13090 via reconstitution of its production in a heterologous host. We demonstrated that TubE specifically utilizes phosphoribosylpyrophosphate and 7-carboxy-7-deazaguanine for the precise construction of the deazapurine nucleoside scaffold. Moreover, we provided biochemical evidence that TubD functions as an NADPH-dependent reductase, catalyzing irreversible reductive deamination. Finally, we verified that TubG acts as a Nudix hydrolase, preferring Co2+ for the maintenance of maximal activity, and is responsible for the tailoring hydrolysis step leading to TBN.ConclusionsThese findings lay a foundation for the rational generation of TBN analogs through synthetic biology strategy, and also open the way for the target-directed search of TBN-related antibiotics.
Highlights
Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, high‐ lights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond
We have identified the TBN biosynthetic gene cluster from S. tubercidicus NBRC 13090 (S. tubercidicus hereafter) by engineered production of TBN in a heterologous host, and have further elucidated that TBN biosynthesis involves a PRPPdependent assembly logic associated with tailoring reduction and phosphohydrolysis steps
Identification of the TBN biosynthetic gene cluster To identify the gene cluster for TBN biosynthesis, the genome of S. tubercidicus was sequenced by an Illumina Hiseq method, rendering appr. 7.88-Mb of non-redundant bases after assembly of clean reads (Additional file 1: Table S1)
Summary
Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, high‐ lights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond. Pyrrolopyrimidine ( known as 7-deazapurine) containing compounds have been discovered to be widely distributed in nature as microbial secondary metabolites (SMs) and as hypermodified base (queuosine) in RNA [1, 2]. This family of SMs shows diverse biological functions, ranging from cofactors (involved in the biosynthesis of tetracycline antibiotics) as well as DNA repair to antibiotics with herbicidal, antibacterial, antiviral, antifungal, antitumor, and antineoplastic. GCH I, GTP cyclohydrolase I (ToyD); QueD/ToyB, 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase; H 2NTP, 7, 8-dihydroneopterin-3′-triphosphate; QueE/ToyC, 7-carboxy-7-deazaguanine (CDG) synthase; QueC/ToyM, 7-cyano-7-deazaguanine (PreQ0) synthetase
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