Aminoglycoside (AG) antibiotics are used to treat many Gram-negative and some Gram-positive infections and, importantly, multidrug-resistant tuberculosis. Among various bacterial species, resistance to AGs arises through a variety of intrinsic and acquired mechanisms. The bacterial cell wall serves as a natural barrier for small molecules such as AGs and may be further fortified via acquired mutations. Efflux pumps work to expel AGs from bacterial cells, and modifications here too may cause further resistance to AGs. Mutations in the ribosomal target of AGs, while rare, also contribute to resistance. Of growing clinical prominence is resistance caused by ribosome methyltransferases. By far the most widespread mechanism of resistance to AGs is the inactivation of these antibiotics by AG-modifying enzymes. We provide here an overview of these mechanisms by which bacteria become resistant to AGs and discuss their prevalence and potential for clinical relevance.

Covering: up to May 2006. This Highlight provides an overview of the molecular logic employed in the formation and elaboration of pyrroles during natural product biosyntheses. The pyrrole ring, either in its unadorned form or as the pyrrole-2-carboxylate moiety commonly encountered in natural products, possesses many useful chemical and electronic features that are readily exploited in biological contexts. The ring itself is planar and electron rich, highly susceptible to electrophilic attack, capable of oxidation and able to participate in both π–π stacking and hydrogen-bonding interactions; the 2-carboxylate moiety provides a nucleophilic center for derivatization, hydrogen bonding and reaction with biological targets. The focus of this Highlight is primarily on the construction of pyrrole rings during secondary metabolite formation, and on their derivatization and incorporation into natural products. Overall biosynthetic strategies are discussed as well as relevant enzymology and mechanistic details.

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of l -Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH , are predicted to encode the flavin reductase and halogenase components of an FADH ₂ -dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O ₂ , chloride ion, and l -Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH ₂ for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k _cat of 1.4 min ^–1 was observed for the RebF/RebH system but increased at least 10-fold in low-pO ₂ conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Background: Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit. Results: We have developed a rapid method of scanning genome sequences for multiple polyketide, nonribosomal peptide, and mixed combination natural products with output in a text format that can be readily converted to two and three dimensional structures using conventional software. Our open-source and web-based program can assemble various small molecules composed of twenty standard amino acids and twenty two other chain-elongation intermediates used in nonribosomal peptide systems, and four acyl-CoA extender units incorporated into polyketides by reading a hidden Markov model of DNA. This process evaluates and selects the substrate specificities along the assembly line of nonribosomal synthetases and modular polyketide synthases. Conclusion: Using this approach we have predicted the structures of natural products from a diverse range of bacteria based on a limited number of signature sequences. In accelerating direct DNA to metabolomic analysis, this method bridges the interface between chemists and biologists and enables rapid scanning for compounds with potential therapeutic value.