A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis in actinomycetes is a prerequisite for engineering production of analogs with enhanced antitumor activity. Several proteins expected to synthesize the PBD’s dihydropyrrole moiety were heterologously expressed, purified and assayed for activity. UV-visible spectroscopy revealed that predicted dioxygenases SibV and homolog Orf12 associated with PBDs sibiromycin and anthramycin, respectively, 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 indicated that L-2,3-secodopa then spontaneously cyclizes into the α-keto acid tautomer of 4-(2-oxo-3-butenoic-acid)-4,5-dehydro-L-proline 1.1 (λmax = 414 nm). Thus, the dioxygenases establish the scaffold of the dihydropyrrole moiety. Both the quaternary structure and product formed by dioxygenases are conserved in dihydropyrrole biosynthesis within both PBD and non-PBD pathways. Stability studies suggest that 1.1 is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps.

Hydrolysis and methylation steps were proposed to modify the dihydropyrrole scaffold in PBDs. The predicted proteins SibS and homolog TomK associated with sibiromycin and PBD tomaymycin biosynthesis, respectively, were assayed for hydrolysis activity. The predicted protein SibZ associated with sibiromycin biosynthesis was assayed for methyltransferase activity. The proposed SibZ substrate 4-vinyl-4,5-dehydro-L-proline 1.2 was synthesized. For these three proteins, no catalytic activity was observed with their proposed substrates or substrate precursors under a range of conditions. However, SibS binds 1.1 (KD = 64 ± 2 µM) suggesting it participates in dihydropyrrole biosynthesis. HPLC-MS indicated that SibS catalyzes the depurination of S-adenosylmethionine although it is unlikely this reaction is involved in sibiromycin biosynthesis. These findings suggest that the pathway requires revision.

The adenylation and thiolation didomain of the predicted non-ribosomal peptide synthetase SibD associated with sibiromycin biosynthesis was expressed and purified. To test if SibD incorporates the dihydropyrrole moiety into sibiromycin, its proposed substrate 4-propenyl-4,5-dehydro-L-proline 1.5 was synthesized. A radioactivity exchange assay and peptide analysis by MS revealed that SibD does not adenylate and thiolate 1.5 or its precursors L-DOPA, 1.1 or 1.2. However, L-threonine and the metabolic precursor L-tyrosine are substrates for these reactions. SibB promotes adenylation catalyzed by SibD and represents one of two proteins, distinct from MbtH-like proteins, capable of promoting adenylation.