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 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 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.     

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.     

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

The presence of β‐branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential β‐branching modifications. We use purified single and multi‐domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of β‐branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of ¹³C NMR of a single ¹³C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl‐CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific β‐branch.     

A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran‐Forming Cyclase

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically‐derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa‐conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran‐forming cyclase from a cis‐AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.     

A Rearrangement of 3‐Pyrazolines as a Missing Link

The thermal conversion of 4‐isoxazolines to 4‐oxazolines involves the transposition of two ring members. The ring‐contraction and ring‐expansion sequence in the reaction 2 → 5 has been previously clarified. The low N−N bond energy should favor an analogous conversion of 3‐pyrazolines 6 to 4‐imidazolines 7 ; the first example of such a transformation is reported here. In the yellow 16 , the 3‐pyrazoline is part of a pyrazolo[5,1‐a]isoquinoline system. Daylight induces a ring contraction, which affords the 2‐isoquinolylaziridine derivative 21 . The latter is converted at 65° to the tricyclic 4‐imidazoline 26 by a sequence of electrocyclic aziridine ring‐opening and 1,5‐electrocyclization of a C=N‐conjugated azomethine ylide 25 .