Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a "modular" strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.

Tomaymycin produced by Streptomyces achromogenes is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.

Pyrrolobenzodiazepines (PBDs) are sequence selective DNA alkylating agents with remarkable antineoplastic activity. They are either naturally produced by actinomycetes or synthetically produced. The remarkable broad spectrum of activities of the naturally produced PBDs encouraged the synthesis of several PBDs, including dimeric and hybrid PBDs yielding to an improvement in the DNA binding sequence specificity and in the potency of this class of compounds. However, limitation in the chemical synthesis prevented the testing of one of the most potent PBDs, sibiromycin, a naturally produced glycosylated PBDs. Only recently the biosynthetic gene clusters for PBDs have been identified opening the doors to the production of glycosylated PBDs by mutasynthesis and biosynthetic engineering. The present review describes the recent studies on the biosynthesis of naturally produced pyrrolobenzodiazepines. In addition, it provides an overview on the isolation and characterization of naturally produced PBDs, on the chemical synthesis of PBDs, on the mechanism of DNA alkylation, and on the DNA binding affinity and cytotoxic properties of both naturally produced and synthetic pyrrolobenzodiazepines.

NAD(+) is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD(+) biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD(+) synthetase, which catalyzes the ATP-dependent formation of NAD(+) at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD(+) synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD(+) synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-A intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

The transition-state structures and mechanisms of the isomerization to the 2‘-isomer and cleavage reactions of uridine 3‘-m-nitrobenzyl phosphate to m-nitrobenzyl alcohol and a 2‘,3‘-cyclic UMP at 86 °C and at pH 2.5, 5.5, and 10.5 have been characterized through kinetic isotope effects. The 18O primary isotope effect of 1.0019 ± 0.0007 and the secondary isotope effect of 0.9904 observed for the cleavage reaction at pH 2.5 are consistent with a neutral phosphorane-like transition-state structure. The cleavage and isomerization reactions at pH 2.5 proceed through a neutral phosphorane intermediate. The 18 k bridge and 18 k nonbridge of unity measured for the pH-independent isomerization reaction at neutral pH support a stepwise mechanism with a monoanionic phosphorane intermediate. The primary and secondary isotope effects of 1.009 ± 0.001 and of 0.9986 ± 0.0004 observed for the pH-independent cleavage reaction are consistent with either a stepwise mechanism through a monoanionic phosphorane intermediate or with an ANDN reaction with a transition state resembling a monoanionic phosphorane intermediate. The absolute requirement of a water-mediated proton transfer for the formation of a phosphorane intermediate is proven by the absence of the isomerization reaction in anhydrous tert-butyl alcohol. The primary isotope effect of 1.0272 ± 0.0001 for the cleavage reaction at pH 10.5 is consistent with a concerted reaction through a transition state in which the leaving group departs with almost a full negative charge.