No abstract

Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

The biosynthesis of many natural products of clinical interest involves large, multi-domain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins co-purify together with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.Nonribosomal peptide synthetases (NRPSs) are involved in the assembly of natural products of clinical interest such as the antibacterial drugs vancomycin, daptomycin, and capreomycin. A basic understanding how NRPSs catalyze the assembly of such molecules from simple precursors has been established (1). During assembly, each precursor is activated, covalently tethered to the NRPS, and then directionally condensed into the growing molecule by a set of catalytic domains grouped together as modules. Each module is typically composed of an adenylation (A) domain that recognizes and activates each precursor and tethers them to a peptidyl carrier protein (PCP) domain as a thioester. Condensation (C) domains subsequently catalyze directional bond formation between two PCP-linked precursors. Additional domains can add functionality to the precursors or govern its release from the NRPS. The repeating domain/modular structure of NRPSs provides an assembly line-like logic to the biosynthesis of the associated natural products.In bacteria, many gene clusters coding for the NRPS involved in the production of natural products also code for a small (∼70 amino acid) protein containing three conserved tryptophan residues. These proteins have been named the MbtH-like protein superfamily based on their similarity to MbtH from the mycobactin biosynthesis gene cluster (2). The production of some NRPS-dependent natural products requires an MbtH-like protein (3,4), but how these proteins influence production is unknown. Co-production of an MbtH-like protein with an NRPS component enhances protein production levels (5). A direct role in catalysis has been questioned by a report that the enterobactin (ENT) NRPS is functional in vitro in the absence of the associated MbtH-like protein (6). Structural work on the MbtHlike protein from the pyroverdine system (3) and MbtH itself (7) did not reveal any motifs suggestive of a catalytic site, instead, a role in protein-protein interactions.We are investigating the biosynthesis of the antituberculosis drugs capreomycin (CMN) and viomycin (VIO) to better understand NRPS enzymology and develop new derivatives of these drugs using combinatorial biosynthesis. These structurally related non-ribosomal * To whom correspondence should be addressed. Phone: (608) Figure S1, Supporting Information) (8, 9). In addition to the NRPS components, the associated gene clusters code for MbtH-like proteins. This provided us with two related NRPS systems to address questions co...

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.antibiotics ͉ combinatorial biosynthesis

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that ϳ50 uncharacterized genes were regulated by changes in light intensity and O 2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H 2 as an electron donor and CO 2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO 2 ) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO 2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO 2 , or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions. Life on earth is dependent upon photosynthesis, either directly using energy from sunlight or indirectly through the use of organic compounds produced by photosynthetic organisms. Due to the importance of photosynthesis, much research has been devoted to understanding this process in plants, algae, and photosynthetic bacteria. The defining feature of all photosynthetic organisms is their ability to use light energy to generate reducing power, which is used to support the synthesis of ATP, the assimilation of CO 2 , or the synthesis of other compounds. We are studying the requirements for photosynthesis in the ␣-proteobacterium Rhodobacter sphaeroides, a facultative phototroph that also can produce energy by aerobic or anaerobic respiratory pathways.Since the continual flow of electrons through electron carriers is critical to energy production, cells have evolved strategies to maintain necessary electron flow by recycling or disposing of excess reducing power. For example, fermentative pathways use organic compounds as electron acceptors, and electron transport to O 2 allows the aerobic respiratory chain to produce energy. When O 2 is absent, anaerobic respiratory pathways reduce alternate electron acceptors like dimethyl sulfoxide (DMSO) to dispose of excess reducing power (18). Photosynthetic growth also requires a strategy for recycling reducing power, but di...