A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1.

Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba.

Biosynthesis of piperazic acid via N5-hydroxy-ornithine in Kutzneria spp. 744.

Which came first? We have investigated the biosynthesis of the piperazic acid (Piz) building blocks in the kutzneride family of metabolites. The flavin-dependent oxygenase KtzI was shown to convert ornithine to N(5)-OH-Orn. LC-MS/MS showed (13)C(5)-labeled versions of these two amino acids to be direct precursors of piperazic acid in vivo.

Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2.

The alpha-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His(2)(Asp/Glu)(1) "facial triad" of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X(-)) coordinates at the vacated site. Halogenation is thought to result from "rebound" of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H(*)) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H(*)-abstraction proficiency for halogen-rebound selectivity. Upon replacement of L-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid L-norvaline, decay of the chloroferryl intermediate becomes 130x faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H(*) abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate --> halide ligand swap combine to specify the halogenation outcome.

Biosynthetic chlorination of the piperazate residue in kutzneride biosynthesis by KthP.

Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,5S)-5-chloropiperazate, distinct from the 3S,5R stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks.

Halogenation strategies in natural product biosynthesis.

Halogenation is a frequent modification of secondary metabolites and can play a significant role in establishing the bioactivity of a compound. Enzymatic halogenation through oxidative mechanisms is the most common route to these metabolites, though direct halogenation via halide anion incorporation is also known to proceed through both enzymatic and nonenzymatic pathways. In this article, we review the current state of knowledge regarding the mechanisms of these transformations, highlight applications of this knowledge, and propose future opportunities and challenges for the field.

Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an Fe(II)-dependent halogenase and a cyclopropane-forming flavoprotein.

The biosynthetic gene cluster for the kutzneride family of hexapeptidolactones includes the four-gene cassette ktzABCD postulated to generate a nonproteinogenic amino acid. Encoded by this cassette are the nonheme FeII-dependent halogenase KtzD and the acyl-CoA dehydrogenase-like flavoprotein KtzA, proposed to work in conjunction with adenylating protein KtzB and carrier protein KtzC. In the present work, we report the in vitro reconstitution of this four-protein system and identify the final product as (1S,2R)-allocoronamic acid bound in thioester linkage to KtzC. Further analysis of KtzD and KtzA support a biosynthetic pathway that involves KtzD-mediated generation of a gamma-chloroisoleucyl intermediate which is cyclized to the final product by KtzA without redox participation of the bound flavin cofactor. This work introduces a new monomer for potential incorporation into nonribosomal peptides and validates the unique strategy for its biosynthesis.

Targeted Delivery of Cytotoxic NAMPT Inhibitors Using Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are a therapeutic modality that enables the targeted delivery of cytotoxic drugs to cancer cells. Identification of active payloads with unique mechanisms of action is a key aim of research efforts in the field. Herein, we report the development of inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) as a novel payload for ADC technology. NAMPT is a component of a salvage biosynthetic pathway for NAD, and inhibition of this enzyme results in disruption of primary cellular metabolism leading to cell death. Through derivatization of the prototypical NAMPT inhibitor FK-866, we identified potent analogues with chemical functionality that enables the synthesis of hydrophilic enzyme-cleavable drug linkers. The resulting ADCs displayed NAD depletion in both cell-based assays and tumor xenografts. Antitumor efficacy is demonstrated in five mouse xenograft models using ADCs directed to indication-specific antigens. In rat toxicology models, a nonbinding control ADC was tolerated at >10-fold the typical efficacious dose used in xenografts. Moderate, reversible hematologic effects were observed with ADCs in rats, but there was no evidence for the retinal and cardiac toxicities reported for small-molecule inhibitors. These findings introduce NAMPT inhibitors as active and well-tolerated payloads for ADCs with promise to improve the therapeutic window of NAMPT inhibition and enable application in clinical settings.

Identification of a eukaryotic reductive dechlorinase and characterization of its mechanism of action on its natural substrate.

Chlorinated compounds are important environmental pollutants whose biodegradation may be limited by inefficient dechlorinating enzymes. Dictyostelium amoebae produce a chlorinated alkyl phenone called DIF which induces stalk cell differentiation during their multicellular development. Here we describe the identification of DIF dechlorinase. DIF dechlorinase is active when expressed in bacteria, and activity is lost from Dictyostelium cells when its gene, drcA, is knocked out. It has a K(m) for DIF of 88 nM and K(cat) of 6.7 s(-1). DrcA is related to glutathione S-transferases, but with a key asparagine-to-cysteine substitution in the catalytic pocket. When this change is reversed, the enzyme reverts to a glutathione S-transferase, thus suggesting a catalytic mechanism. DrcA offers new possibilities for the rational design of bioremediation strategies.

Cloning and characterization of the biosynthetic gene cluster for kutznerides.

Kutznerides, actinomycete-derived cyclic depsipetides, consist of six nonproteinogenic residues, including a highly oxygenated tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1-methylcyclopropyl)-glycine, a beta-branched-hydroxy acid, and 3-hydroxy glutamic acid, for which biosynthetic logic has not been elucidated. Herein we describe the biosynthetic gene cluster for the kutzneride family, identified by degenerate primer PCR for halogenating enzymes postulated to be involved in biosyntheses of these unusual monomers. The 56-kb gene cluster encodes a series of six nonribosomal peptide synthetase (NRPS) modules distributed over three proteins and a variety of tailoring enzymes, including both mononuclear nonheme iron and two flavin-dependent halogenases, and an array of oxygen transfer catalysts. The sequence and organization of NRPS genes support incorporation of the unusual monomer units into the densely functionalized scaffold of kutznerides. Our work provides insight into the formation of this intriguing class of compounds and provides a foundation for elucidating the timing and mechanisms of their biosynthesis.

A library of spirooxindoles based on a stereoselective three-component coupling reaction.

A collection of structurally complex and chemically diverse small molecules is a useful tool to explore cell circuitry. In this article, we report the split-pool synthesis of more than 3000 spirooxindoles on high capacity macrobeads. The key reaction to assemble the spirooxindole core stereoselectively is a Lewis acid variant of the Williams' three-component coupling. After formation, the skeleton was elaborated using Sonogashira couplings, amide forming reactions, and N-acylations of gamma-lactams. The final library was analyzed by sampling individual macrobeads and by using binomial confidence limits. It was determined that at least 82% of the library compounds should have better than 80% purity. To demonstrate the utility of our discovery process, a high-throughput chemical genetic modifier screen was performed using stock solutions of the resultant products. A number of positives were identified as enhancers of the cellular actions of latrunculin B, an actin polymerization inhibitor. Through resynthesis, we confirmed one of the positives and demonstrated that, in yeast cells, it has an EC50 in the sub-micromolar range.