A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of L-3,4-dihydroxyphenylalanine (L-DOPA) to form L-2,3-secodopa (λmax = 368 nm). 1H NMR spectroscopy indicates that L-2,3-secodopa cyclizes into the α-keto acid tautomer of L-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax = 414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole or pyrrolidine biosynthesis within both PBD and non-PBD pathways.

Nonribosomal peptide synthetases (NRPSs), responsible for synthesizing many medicinally important natural products, frequently use adenylation domain activators (ADAs) to promote substrate loading. Although ADAs are usually MbtH-like proteins (MLPs), a new type of ADA appears to promote an NRPS-dependent incorporation of a dihydropyrrole unit into sibiromycin. The adenylation and thiolation didomain of the NRPS SibD catalyzes the adenylation of a limited number of amino acids including L-tyr, the precursor in dihydropyrrole biosynthesis, using a standard radioactivity exchange assay. LC-MS/MS analysis confirmed loading of L-tyr onto the thiolation domain. SibB, a small protein with no prior functional assignment nor sequence homology to MLPs, was found to promote the exchange activity. MLPs from bacteria expressing homologous biosynthetic pathways were unable to replace this function of SibB. Discovery of this new type of ADA demonstrates the importance of searching beyond the conventional MLP standard for proteins affecting NRPS activity. Graphical AbstractNonribosomal peptide synthetases (NRPSs) frequently use MbtH-like proteins to promote activity of their adenylation domains. Sibiromycin biosynthesis provides an example of a pathway that lacks such a protein and instead uses an atypical adenylation domain activator, SibB, to promote the activity of the NRPS SibD.Correspondence to: Shalini Saha; Steven E. Rokita. Experimental SectionAll experimental details including the construction of expression vectors, production and purification of proteins, synthesis and characterization of all compounds, and assays for radioisotope exchange and NRPS acylation are described in supporting information. Elongation modules include an additional condensation (C) domain for coupling two NRPSconjugates through a peptide bond. Termination modules contain yet another domain most typically a thioesterase (TE) domain or infrequently a reductase (R) domain that promotes hydrolysis of the nascent peptide from the NRPS. These modules often are complemented by a protein known as an A domain activator (ADA). [2] ADAs are small proteins that stimulate A domain activity and were first found necessary for successful biosynthesis of capreomycin and viomycin. [3] In a few cases, NRPSs were not even observed or isolated in a soluble form after heterologous expression without coexpression of an appropriate ADA. [4] The first ADAs reported were all MbtH-like proteins (MLPs) named after a homolog (MbtH) in Mycobacterium tuberculosis and identifiable by a Pfam domain (PF03621). [5] More recently, two new ADAs have been discovered that are devoid of the MLP signature sequences. One of these resembles an incomplete C domain and the other shares very low sequence similarity to known NRPSs (E > 0.002). [6] Both of these ADAs as well as one example of an MLP are covalently fused to their A domains. [6][7] In contrast, the vast majority of ADAs exist as independent proteins encoded by genes within their associated biosynthetic gene cluster...

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