Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β′ bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the β′ F-loop and the β fork loop. Collectively, our data are consistent with a model in which the β subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the β subunit.

Anthracyclines are important anticancer drugs. We discuss recent insights into the biosynthetic pathways and bioactivities of anthracyclines, and evaluate the discovery and engineering of effective derivatives with less severe side effects.

Microbial natural products are an important source of chemical entities for drug discovery. Recent advances in understanding the biosynthesis of secondary metabolites has revealed how this rich chemical diversity is generated through functional differentiation of biosynthetic enzymes. For instance, investigations into anthracycline anticancer agents have uncovered distinct S-adenosyl methionine (SAM)-dependent proteins: DnrK is a 4-O-methyltransferase involved in daunorubicin biosynthesis, whereas RdmB (52% sequence identity) from the rhodomycin pathway catalyzes 10-hydroxylation. Here, we have mined unknown anthracycline gene clusters and discovered a third protein subclass catalyzing 10-decarboxylation. Subsequent isolation of komodoquinone B from two Streptomyces strains verified the biological relevance of the decarboxylation activity. Phylogenetic analysis inferred two independent routes for the conversion of methyltransferases into hydroxylases, with a two-step process involving loss-of-methylation and gain-of-hydroxylation presented here. Finally, we show that simultaneously with the functional differentiation, the evolutionary process has led to alterations in substrate specificities.

SnoaB is a cofactor-independent monooxygenase that catalyzes the conversion of 12-deoxynogalonic acid to nogalonic acid in the biosynthesis of the aromatic polyketide nogalamycin in Streptomyces nogalater. In vitro (18)O(2) experiments establish that the oxygen atom incorporated into the substrate is derived from molecular oxygen. The crystal structure of the enzyme was determined in two different space groups to 1.7 and 1.9 A resolution, respectively. The enzyme displays the ferredoxin fold, with the characteristic beta-strand exchange at the dimer interface. The crystal structures reveal a putative catalytic triad involving two asparagine residues, Asn18 and Asn63, and a water molecule, which may play important roles in the enzymatic reaction. Site-directed mutagenesis experiments, replacing the two asparagines individually by alanine, led to a 100-fold drop in enzymatic activity. Replacement of an invariant tryptophan residue in the active site of the enzyme by phenylalanine also resulted in an enzyme variant with about 1% residual activity. Taken together, our findings are most consistent with a carbanion mechanism where the deprotonated substrate reacts with molecular oxygen via one electron transfer and formation of a caged radical.