1. Volume 24, Issue 2, Pages 171-181 (February 2017)
An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics 
Pan Wu, Dan Wan, Gudan Xu, Gui Wang, Hongmin Ma, Tingting Wang, Yaojie Gao, Jianzhao Qi, Xiaoxia Chen, Jian Zhu, Yong-Quan Li, Zixin Deng, Wenqing Chen 
Cell Chemical Biology 
Volume 24, Issue 2, Pages (February 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 171-181DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1
Chemical Structures of PTN, Ara-A, and Related Nucleoside Antibiotics
(A) Chemical structures of PTN and Ara-A and related antibiotic pairs. The antibiotic on top and the corresponding one below it constitute a pair concomitantly produced by a single microorganism. The PTN and Ara-A (or PTN and cordycepin) pair is concomitantly produced by S. antibioticus and Aspergillus nidulans Y176-2, respectively; the coformycin and formycin pair is produced by Streptomyces kaniharaensis ATCC and Nocardia interforma ATCC The 2′-Cl pentostatin (2′-Cl PTN) and 2′-amino-2′-deoxyadenosine (2′-Amino dA) pair is produced by Actinomudura sp. ATCC
(B) Chemical structures of Ara-U, Ara-T, Ara-C, and Ara-I.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Genetic Organization and Verification of the pen Gene Cluster
(A) Genetic organization of the pen gene cluster.
(B) HPLC profiles of the metabolites produced by wild-type and the TD3 mutant of S. antibioticus. ST, authentic standard mixture of PTN, Ara-A, and Ara-I; WT, metabolites from wild-type S. antibioticus; TD3, metabolites from the TD3 mutant of S. antibioticus. The HPLC profiles of PTN, Ara-I, and Ara-A at specific retention time are highlighted in the shaded regions.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
Genetic Investigation of the PTN and Ara-A Biosynthetic Pathways
(A) Extract ion chromatography (EIC) analysis of the metabolites produced by S. aureochromogenes CXR14::pWHU1106 and its variants. ΔpenA is the sample from the strain of S. aureochromogenes CXR14 containing pWHU1106/ΔpenA, and other related samples are correspondingly assigned. ΔpenHI refers to the sample from S. aureochromogenes CXR14 containing pWHU1106/ΔpenHI, from which penHI was simultaneously in-frame deleted via a PCR targeting strategy.
(B) EIC analysis of the samples of ΔpenB and ΔpenC recombinants. The highlighted rectangular region is enlarged to show the characteristic [M + H]+ ion of Ara-I as indicated in the right column.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
In Vitro Characterization of PenB as a Reversible Oxidoreductase
(A) SDS-PAGE analysis of the purified oxidoreductase PenB.
(B) Schematic of the PenB-catalyzed reversible reaction in the presence of the redox cofactor NAD(P)+ or NAD(P)H.
(C) LC-MS analysis of the extracted ion chromatogram (EIC) for the reverse reaction of PenB using PTN as substrate. (i) EIC analysis of the PenB reaction using NADP+ as cofactor; (ii) EIC analysis of the PenB reaction using NAD+ as cofactor; (iii) EIC analysis of the PenB reaction without cofactor added, as negative control; (iv) EIC analysis of the reaction without PenB (but with NADP+ added), as negative control.
(D) LC-MS analysis of the forward reaction of PenB using 6-keto PTN as substrate. (i) EIC analysis of the PenB reaction using NADPH as cofactor; (ii) EIC analysis of the PenB reaction using NADH as cofactor; (iii) EIC analysis of the PenB reaction without cofactor added, as negative control; (iv) EIC analysis of the reaction without PenB (but with NADPH added), as negative control.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Biochemical Characterization of SanADA3 as the Adenosine/Ara-A Deaminase and PTN as the Deaminase Inhibitor
(A) SDS-PAGE analysis of S. antibioticus adenosine deaminase 3 (SanADA3, abbreviated as ADA3).
(B) Schematic of the SanADA3-catalyzed reaction. PTN functions as a potent inhibitor of SanADA3.
(C) HPLC traces of the SanADA3-catalyzed reaction with adenosine as substrate and PTN as competitive inhibitor. (i) The authentic standard of inosine; (ii) ADA3 reaction without PTN added; (iii) ADA3 reaction with 1 mM PTN (final conc.) added; (iv) ADA3 reaction with 0.1 mM PTN (final conc.) added; (v) ADA3 reaction with 0.01 mM PTN (final conc.) added; (vi) negative control without enzyme added.
(D) HPLC traces of the SanADA3-catalyzed reaction with Ara-A as substrate and PTN as inhibitor. (i) The authentic standard of Ara-I; (ii) ADA3 reaction without PTN added; (iii) ADA3 reaction with 1 mM PTN (final conc.) added; (iv) ADA3 reaction with 0.1 mM PTN (final conc.) added; (v) ADA3 reaction with 0.01 mM PTN (final conc.) added; (vi) negative control without enzyme added.
(E) Michaelis-Menten plots of inhibition of SanADA3 activity by PTN with different concentrations of adenosine as substrate. KM (18 ± 3 μM) and kcat (135 ± 13 s−1) were measured with no inhibitor PTN added. The inhibition constant (Ki =  ±  μM) was measured with different concentrations of PTN (1, 2, and 5 nM).
(F) Michaelis-Menten plots of inhibition of SanADA3 activity by PTN with different concentrations of Ara-A as substrate. Measurement of KM (51 ± 3 μM) and kcat (37 ± 10 s−1) was carried out with no inhibitor PTN added. The inhibition constant (Ki = 0.001 ±  μM) was measured with different concentrations of PTN (1, 2, and 5 nM). The error bars represent the SD from three different experiments.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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8. Figure 6 Proposed Pathways for PTN and Ara-A Biosynthesis
(A) The proposed pathway for PTN biosynthesis. PRPP, phosphoribosyl pyrophosphate.
(B) The proposed pathway for Ara-A biosynthesis.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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