Volume 24, Issue 10, Pages e8 (October 2017)

Volume 24, Issue 10, Pages 1216-1227.e8 (October 2017)
Total Biosynthesis of the Pyrrolo[4,2]benzodiazepine Scaffold Tomaymycin on an In Vitro Reconstituted NRPS System Alexander von Tesmar, Michael Hoffmann, Jan Pippel, Antoine Abou Fayad, Stefan Dausend-Werner, Armin Bauer, Wulf Blankenfeldt, Rolf Müller Cell Chemical Biology Volume 24, Issue 10, Pages e8 (October 2017) DOI: /j.chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 1216-1227. e8DOI: (10. 1016/j. chembiol
Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Chemical Structures of the Fourteen Tested Anthranilic Acids as well as the Five Tested Proline Derivatives, Including a Schematic Overview of the Applied Methodology Derivatives, which could serve as potential substrates based on the results of the respective methods are highlighted with colored frames. Based on in vitro loading assays with TomA and subsequent LC-MS measurements on intact protein level, ten derivatives would be conceivable (blue frames). Only incorporation of five derivatives could be observed by performing in vitro reconstitution assays with respective LC-MS measurements on small-molecule level (yellow frames). In vitro assays with TomG with respective small-molecule analytics showed that only one derivative is processed (purple frames). Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Steps of the Tomaymycin In Vitro Reconstitution Including the Respective Deconvoluted Mass Spectra for Each Protein Species to Confirm the Substrate Binding and thus Monitoring the Complete Biosynthesis The observed mass deviation in the case of the dipeptide-loaded TomB is attributable to overlapping MS signals of the occurring species due to incomplete conversion. Hence, the limits of maximum entropy deconvolution algorithm is reached leading to inaccurate mass detection. Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Scheme Showing the Workflow of TomA Loading Assays, Including the Results of Loading Attempts with Different Available Anthranilic Acid Derivatives The top panel shows exemplary LC-MS results obtained for loading assays of TomA. Charge state distributions were typically found to be in the range of 800–1,200 m/z and deconvoluted mass spectra allowed to obtain the respective protein mass typically within <10 ppm. Mass shifts shown in the lower panel are given as delta masses with respect to holo TomA. Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Approaches toward the Analysis of Substrate Specificity of TomA by Competitive Loading Assays with Pairs of Anthranilic Acid Derivatives in Equimolar Ratio Detected delta masses are related to holo TomA and showed which substrate is preferred. Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Structural Analysis Identifies TomG as Class I O-Methyltransferase (A) Overview of the dimeric crystal structure of TomG (monomer A, yellow and orange; monomer B, blue) with the bound cofactor S-adenosyl methionine (SAM) (stick presentation in green). (B) Proposed binding modes of 4,5-dihydroxy anthranilic acid (4,5-dihyd-AA; in light gray) and 4,5-dihydroxy-PBD (4,5-dihyd-PBD; two binding modes in stick presentation in light and dark blue) to the TomG monomer (Mg2+ ion shown as orange sphere; molecular surface colored by electrostatic potential); figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version Schrödinger, LLC). Secondary structure elements were assigned with the aid of the STRIDE webserver (Frishman and Argos, 1995). Electrostatic potential was calculated using the APBS 2.1 plugin (Baker et al., 2001) under PyMOL and the PDB2PQR webserver ( Dolinsky et al., 2007). Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Tomaymycin Biosynthesis Comprising Hydroxylation of 5-OH Anthranilic Acid by TomE and TomF, Methylation by TomG, and Subsequent PBD Assembly by TomAB According to our data, tailoring of anthranilic acid likely takes place either online while tethered to TomA or prior to NRPS assembly. Cell Chemical Biology , e8DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions

Volume 24, Issue 10, Pages e8 (October 2017)

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Volume 24, Issue 10, Pages e8 (October 2017)

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