Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

A Long‐Range Acting Dehydratase Domain as the Missing Link for C17‐Dehydration in Iso‐Migrastatin Biosynthesis

The dehydratase domains (DHs) of the iso‐migrastatin (iso‐MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module‐6, ‐9, ‐10 of MgsF (i.e., DH6, DH9, DH10) and module‐11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso‐MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β‐hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long‐range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.     

Biosynthesis of the Carbonylmethylene Structure Found in the Ketomemicin Class of Pseudotripeptides

We recently discovered novel pseudotripeptides, the ketomemicins, which possess a C‐terminal pseudodipeptide connected with a carbonylmethylene instead of an amide bond, through heterologous expression of gene clusters identified in actinobacteria. The carbonylmethylene structure is a stable isostere of the amide bond and its biological significance has been shown in several natural and synthetic products. Despite the biological importance of these compounds, little is known about how the carbonylmethylene structure is biosynthesized. In this work, we fully characterized the biosynthetic machinery of the pseudodipeptide. An aldolase, dehydratase, PLP‐dependent glycine‐C‐acetyltransferase, and dehydrogenase were involved in the formation of the pseudodipeptide, with malonyl‐CoA and phenylpyruvate as starter substrates.     

Synthesis,Structural Reassignment,and Antibacterial Evaluation of 2,18‐Seco‐Lankacidinol B

Lankacidins are a group of polyketide natural products with activity against several strains of Gram‐positive bacteria. We developed a route to stereochemically diverse variants of 2,18‐seco‐lankacidinol B and found that the stereochemical assignment at C4 requires revision. This has interesting implications for the biosynthesis of natural products of the lankacidin class, all of which possessed uniform stereochemistry prior to this finding. We have evaluated 2,18‐seco‐lankacidinol B and three stereochemical derivatives against a panel of pathogenic Gram‐positive and Gram‐negative bacteria.     

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l ‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l ‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.     

15‐Hydroperoxy‐PGE2: Intermediate in Mammalian and Algal Prostaglandin Biosynthesis

Arachidonic‐acid‐derived prostaglandins (PGs), specifically PGE₂, play a central role in inflammation and numerous immunological reactions. The enzymes of PGE₂ biosynthesis are important pharmacological targets for anti‐inflammatory drugs. Besides mammals, certain edible marine algae possess a comprehensive repertoire of bioactive arachidonic‐acid‐derived oxylipins including PGs that may account for food poisoning. Described here is the analysis of PGE₂ biosynthesis in the red macroalga Gracilaria vermiculophylla that led to the identification of 15‐hydroperoxy‐PGE₂, a novel precursor of PGE₂ and 15‐keto‐PGE₂. Interestingly, this novel precursor is also produced in human macrophages where it represents a key metabolite in an alternative biosynthetic PGE₂ pathway in addition to the well‐established arachidonic acid‐PGG₂‐PGH₂‐PGE₂ route. This alternative pathway of mammalian PGE₂ biosynthesis may open novel opportunities to intervene with inflammation‐related diseases.     

Asymmetric Chemoenzymatic Synthesis of (−)‐Podophyllotoxin and Related Aryltetralin Lignans

(?)‐Podophyllotoxin is one of the most potent microtubule depolymerizing agents and has served as an important lead compound in antineoplastic drug discovery. Reported here is a short chemoenzymatic total synthesis of (?)‐podophyllotoxin and related aryltetralin lignans. Vital to this approach is the use of an enzymatic oxidative C?C coupling reaction to construct the tetracyclic core of the natural product in a diastereoselective fashion. This strategy allows gram‐scale access to (?)‐deoxypodophyllotoxin and is readily adaptable to the preparation of related aryltetralin lignans.     

A Cascade of Redox Reactions Generates Complexity in the Biosynthesis of the Protein Phosphatase‐2 Inhibitor Rubratoxin A

Redox modifications are key complexity‐generating steps in the biosynthesis of natural products. The unique structure of rubratoxin A ( 1 ), many of which arise through redox modifications, make it a nanomolar inhibitor of protein phosphatase 2A (PP2A). We identified the biosynthetic pathway of 1 and completely mapped the enzymatic sequence of redox reactions starting from the nonadride 5 . Six redox enzymes are involved, including four α‐ketoglutarate‐ and iron(II)‐dependent dioxygenases that hydroxylate four sp³ carbons; one flavin‐dependent dehydrogenase that is involved in formation of the unsaturated lactone; and the ferric‐reductase‐like enzyme RbtH, which regioselectively reduces one of the maleic anhydride moieties in rubratoxin B to the γ‐hydroxybutenolide that is critical for PP2A inhibition. RbtH is proposed to perform sequential single‐electron reductions of the maleic anhydride using electrons derived from NADH and transferred through a ferredoxin and ferredoxin reductase pair.     

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

Post‐translational modifying enzymes from the S‐adenosyl‐l ‐methionine (AdoMet) radical superfamily garner attention due to their ability to accomplish challenging biochemical reactions. Among them, a family of AdoMet radical epimerases catalyze irreversible l ‐ to d ‐amino acid transformations of diverse residues, including 18 sites in the complex sponge‐derived polytheonamide toxins. Herein, the in vitro activity of the model epimerase OspD is reported and its catalytic mechanism and substrate flexibility is investigated. The wild‐type enzyme was capable of leader‐independent epimerization of not only the stand‐alone core peptide, but also truncated and cyclic core variants. Introduction of d ‐amino acids can drastically alter the stability, structure, and activity of peptides; thus, epimerases offer opportunities in peptide bioengineering.