1. Volume 23, Issue 8, Pages 935-944 (August 2016)
Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation 
Jianzhao Qi, Dan Wan, Hongmin Ma, Yuanzhen Liu, Rong Gong, Xudong Qu, Yuhui Sun, Zixin Deng, Wenqing Chen 
Cell Chemical Biology 
Volume 23, Issue 8, Pages (August 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 935-944DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1
The Structure, Gene Cluster, and Revised CPOAA Pathway of Polyoxin
(A) Chemical structures of polyoxins; there are 11 biologically active components of polyoxin, and the 2-amino-3,4-diol carboxylate fragment is highlighted in the shaded region.
(B) The related 2-amino-3,4-diol carboxylate fragment containing natural products, sphingofungin C (antifungal) and myriocin (antifungal); both contain 3,4-diepipolyoxamic acid headgroups (shaded region).
(C) The genetic organization of the polyoxin biosynthetic gene cluster; the genes in red are required for CPOAA biosynthesis, and the corresponding previously annotated functions of the target enzymes are described below.
(D) The revised pathway for CPOAA biosynthesis. CAP, carbamoyl phosphate.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Engineered Production of CPOAA in S. lividans TK24
(A) Representational map for the illustration of the engineering production of CPOAA (9) by the strain QW1. PermE* represents the mutated PermE promoter with enhanced activity.
(B) HRMS analysis of the extracted ion chromatogram (EIC) of the metabolites produced by related strains, QW1: S. lividans TK24 derivative containing pWHU1084 and pWHU1064, TK24::pWHU1064: S. lividans TK24 derivative containing pWHU1064 as negative control, and TK24::pIB139: S. lividans TK24 derivative containing pIB139 as negative control.
(C) HRMS analysis of the target ion from QW1 metabolites.
(D) HRMS/MS analysis of the fragment ions for the target QW1 metabolite.
(E) Theoretical fragmentation pattern of 9.
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5. Figure 3
In Vitro Characterizations of PolM as an NADPH-Dependent Reductase
(A) Scheme of the ArgB-catalyzed reaction as well as the formation of N-acetyl-γ-glutamyl hydroxamate (10).
(B) LC-MS analysis of the PolM-catalyzed reactions. Negative control, the reaction without enzyme added; ArgB reaction + NH2OH, NH2OH was added at the end of the ArgB-catalyzed reaction to trap 2 as the stable oxime 10; ArgB + ArgC, the ArgB reaction plus ArgC and NADPH added; ArgB + ArgC(+PolM), the ArgB reaction plus ArgC and NADPH added, with further addition of PolM after the reaction proceeded for 2 hr; ArgB + PolM, the ArgB reaction plus PolM and NADPH added; ST, the authentic standard of 5.
(C) Proposed mechanism for the PolM-catalyzed reaction.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
In Vitro Characterizations of PolN as a Bifunctional Amino Acid N-Acetyltransferase
(A) LC-MS analysis of PolN reaction with 1 and acetyl-CoA as substrates. Negative control, the reaction without enzyme added; PolN reaction, the reaction with PolN added; ST, the authentic standard of 2.
(B) Enzymatic assay of PolN and its variants. NC, the reaction without enzyme added as negative control; WT, the reaction of the wild-type PolN; S22A, the reaction of the PolN S22A variant; the other reactions are as described for S22A. The error bars represent the SD from three different experiments.
(C) Identification of the acetylation site on PolN by HRMS analysis. PolN + Ac-CoA, the sample of PolN reacted with acetyl-CoA; PolN, the sample of PolN only. Red star indicates the target ion for the peptide fragment with the conserved serine residue acetylated.
(D) LC-MS analysis of PolN reaction with 1 and 5 as substrates. Negative control, the reaction without enzyme added; PolN reaction (Glu−), the reaction with PolN added but lacking 1; PolN reaction (Glu+), the reaction with PolN and 1, ST, the authentic standard of 6.
(E) Proposed mechanism for the acetylation and deacetylation catalyzed by PolN.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
HRMS Analysis of 7 Accumulated in CXR14::4620/ΔpolL Metabolites
(A) EIC traces of the metabolites produced by CXR14::4620/ΔpolL strain.
(B) HRMS/MS analysis of the potential 7 from the metabolites produced by CXR14::4620/ΔpolL strain.
(C) HRMS/MS analysis of the authentic standard of 7.
(D) Theoretical fragmentation pattern of the authentic standard of 7.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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8. Figure 6
In Vitro Characterization of PolL and Its Variants, and a Structure Model for PolL
(A) In vitro characterization of PolL as a Fe(II)-dependent, α-KG dioxygenase. Complete reaction, the reaction with 7, α-KG, Fe(II), DTT, and recombinant PolL added; No α-KG, the reaction without α-KG added; No Fe(II), the reaction without Fe(II) added; No Fe(II) + EDTA, the reaction without Fe(II) but with EDTA added; No DTT, the reaction without DTT added; Nitrogen atmosphere, the complete reaction was incubated under nitrogen atmosphere; ST, the authentic standard of CPOAA (9). Except the control group of nitrogen atmosphere, other reactions were carried out under aerobic conditions.
(B) The homology structure model of PolL. This model is constructed based on Mpe_A2762 (3PL0) from Methylibium petroleiphilum, and the active sites are defined in the rectangular region. The key conserved residues (H163XD165X51H216) involved in binding Fe(II) and α-KG are correspondingly indicated in the amplified rectangular region (right). The dotted lines indicate the interaction force between two related atoms.
(C) LC-MS analysis of the reactions of PolL and its variants. Negative control, reaction without PolL added; PolL reaction, reaction with PolL added for 30 min or 8 hr; ST, the authentic standard of CPOAA. H163A, D165A, and H216A are the individual reaction of PolL variants.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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