Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation

Jing, Qi; Wan, Dan; Ma, Hongmin; Liu, Yuanzhen; Gong, Rong; Qu, Xudong; Sun, Yuhui; Deng, Zixin; Chen, Wenqing

doi:10.1016/j.chembiol.2016.07.011

Cited by 27 publications

(36 citation statements)

References 36 publications

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“…To achieve engineered production of 2=-Cl PTN and 2=-amino dA in a heterologous host, a positive cosmid, 3G12, housing the probable complete ada gene cluster, was screened from the genomic library of Actinomadura sp. ATCC 39365 by a narrowdown PCR strategy (16) and introduced into the heterologous host Streptomyces aureochromogenes CXR14 (17). Subsequently, the recombinant strain (CXR14::3G12), confirmed by PCR, was fermented for further metabolic analysis, and the LC-MS results indicated that the targeted [MϩH] ϩ ions of 2=-Cl PTN (m/z 303.0825), PTN (m/z 269.1220), and 2=-amino dA (m/z 267.1175) could be clearly detected from the sample of the CXR14::3G12 recombinant.…”

Section: Resultsmentioning

confidence: 99%

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

Gao

et al. 2017

Appl Environ Microbiol

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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).

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Section: Resultsmentioning

confidence: 99%

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

Gao

et al. 2017

Appl Environ Microbiol

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“…POLs, a group of structurally related peptidyl nucleoside antibiotics, are produced by Streptomyces cacaoi var. asoensis and Streptomyces aureochromogenes (5). Mechanistically, POL mimics the structure of UDP-N-acetylglucosamine, a building block for fungal chitin biosynthesis.…”

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confidence: 99%

“…1A), and genes polBADHIJK were identified for nucleoside skeleton ( Fig. 1A) formation (5,9). The CPOAA biosynthetic pathway contains an unusual acetylation cycle associated with tandem reduction and sequential hydroxylation (5).…”

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confidence: 99%

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Gong

Jing

et al. 2018

Appl Environ Microbiol

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Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics. POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

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“…A similar outcome could also be observed when α‐amino‐δ‐carbamoylhydroxyvaleric acid was employed as a substrate. Prior to this work, an Fe/αKG from polyoxin biosynthesis, PolL, was previously characterized as a α‐amino‐δ‐carbamoylhydroxyvaleric acid 4‐hydroxylase . However, this enzyme is classified as a member of the PF10014 family, shares only minimal sequence identity with GetI, and affords product with an opposite stereochemical configuration at C4 to GetI.…”

Section: Figurementioning

confidence: 99%

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

2019

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The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

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Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation

Cited by 27 publications

References 36 publications

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

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

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

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