In this issue

Alkaloid engineering by another root

Most metabolic engineering efforts to make natural and 'unnatural' products have focused on bacteria and yeast. However, sparse knowledge about biosynthetic pathways has hindered efforts to recreate alkaloid pathways in microbes. Runguphan and O'Connor have now used genetic engineering approaches to create analogs of alkaloids directly in plant culture. The authors first transformed a previously engineered alkaloid biosynthetic gene with relaxed substrate specificity into Madagascar periwinkle. When commercially available tryptamine analogs were fed to hairy root cultures of the engineered periwinkle plant, the authors observed the production of a variety of unnatural alkaloids. These results highlight the potential for natural product engineering in plants and provide an opportunity to gain new insights into alkaloid biosynthetic pathways. [Brief Communications, p. 151 ; News & Views, p. 140 ] JK

Quieting Shh

The Hedgehog signaling pathway plays a critical role in cancer and development. In this pathway, the N-terminal fragment of Sonic hedgehog (Shh) binds to the transmembrane receptor Patched. This binding event alleviates Patched-mediated repression of the receptor Smoothened, leading to the activation of Gli transcription factors. Current agonists and antagonists of Hedgehog signaling all act at Smoothened or further downstream in the pathway. To identify modulators with a distinct mode of action, Stanton et al. screened a small-molecule microarray, which included 10,000 diversity oriented synthesis compounds and natural products, for small molecules that bound to the N-terminal fragment of Shh. One of the hits, robotnikinin, inhibited Hedgehog signaling upstream of Smoothened in primary human cells and a synthetic model of human skin. [Brief Communications, p. 154 ] JK

Activating fatty acids

Fatty acids are typically activated by fatty acyl-coenzyme A (CoA) ligases (FACLs) that catalyze the formation of an acyladenylate intermediate that is subsequently converted to the corresponding CoA derivative. In contrast, Mycobacterium tuberculosis (Mtb) contains fatty acyl-AMP ligases (FAALs) that only convert fatty acids to acyladenylate derivatives. Arora et al. have solved the first FAAL structure, which revealed a 22-amino-acid insertion common to FAALs but absent from FACLs. Incorporation of this 22-amino-acid stretch into a FACL resulted in the release of acyladenylate derivatives into solution. In contrast, deleting this insertion region from a FAAL enzyme resulted in a gain of FACL activity. To explore the potential of this class of enzymes as an antibacterial target, the authors synthesized acylfulfamoyl analogs that inhibited FAALs, disrupted lipid biosynthesis and reduced cell viability. This study provide a foundation for understanding the biosynthesis and biology of Mtb fatty acids. [Articles, p. 166 ] JK

Achromobactin beginnings

Achromobactin is synthesized in vivo via an NRPS-independent siderophore (NIS) pathway that includes the synthetase AcsD. This enzyme has been proposed to catalyze the condensation of citric acid and ethanolamine to initiate achromobactin synthesis, but the details of this reaction are unknown. Schmelz et al. now demonstrate that the mechanism proceeds with the nucleophilic addition of L-serine to an activated, adenylated citrate. Experiments using ¹³C-labeled citric acid further established the stereochemical course of the reaction. The crystal structure of the enzyme reveals a novel fold, with additional substrate-protein co-complexes demonstrating the basis of AcsD's selectivity. Surprisingly, though the connection between these substructures in the final molecule is through the serine hydroxyl, the O-linked intermediate rearranges in the reaction conditions used to yield an N-linked structure. These results set the stage for investigating additional steps in the achromobactin pathway, particularly as to how subsequent enzymes prevent this O-to-N migration. [Articles, p.174 ; News & Views, p. 143 ] CG

Golgi chemical probe GEFs specific

ADP ribosylating factor 1 (Arf1) is a Golgi-localized GTPase that regulates transport vesicle formation and release. A family of guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP, providing a mechanism for spatiotemporal control of Arf1 activity. Using a cell-based screen, Sáenz et al. identified golgicide A, a small molecule that disrupted the Golgi and trans-Golgi network (TGN). The effects were similar to those of brefeldin A, a broad inhibitor of Arf1 activation, but were shown to be specific for GBF1, a cis-Golgi–localized ArfGEF. Mutagenesis and modeling studies suggest that golgicide A binds in the same cleft between Arf1 and its ArfGEFs as brefeldin A, but forms additional contacts that result in specificity for GBF1. Cellular studies with golgicide A revealed specific roles for GBF1 in maintaining Golgi structure and in traffic through the Golgi and TGN. Golgicide A provides a tool to further dissect the roles of GBF1 in the Golgi and a model for developing additional GEF-specific chemical probes. [Articles, p. 157 ] JK

TRPA1 takes two tasks

Although zinc is essential for the function of hundreds of proteins, high concentrations are cytotoxic. Hu et al. now show that some of the symptoms of overexposure to zinc, including pain, are mediated by the cation channel TRPA1. The authors found that Zn²⁺ acts as a novel second messenger that first enters somatosensory neurons through TRPA1 and then activates the channel through an interaction with several intracellular cysteine and histidine residues. TRPA1 had been previously implicated as a receptor for several pungent phytochemicals, as well as noxious cold. This study now suggests both a Zn²⁺ channel function and a Zn²⁺ sensor function for TRPA1 and adds zinc to the list of other signaling molecules that help modulate sensory transmission. [Articles, p. 183 ; News & Views, p. 141 ] MB

Written by Mirella Bucci, Catherine Goodman & Joanne Kotz