An Enzymatic Cyclopentyl[<i>b</i>]indole Formation Involved in Scytonemin Biosynthesis

An Enzymatic Cyclopentyl[b]indole Formation Involved in Scytonemin Biosynthesi

α,β-Unsaturated β-Silyl Imide Substrates for Catalytic, Enantioselective Conjugate Additions: A Total Synthesis of (+)-Lactacystin and the Discovery of a New Proteasome Inhibitor

Chiral (salen)Al μ-oxo dimer 1 catalyzes the highly enantioselective conjugate addition of carbon-centered nucleophiles to α,β-unsaturated silyl imides. Allyldimethylsilane-substituted imide 4 was identified as an optimal substrate, undergoing addition reactions with a variety of nitrile nucleophiles in high yield and enantiomeric excess. The silicon-containing products are synthetically useful chiral building blocks, as demonstrated by their application to an enantioselective total synthesis of the potent proteasome inhibitor (+)-lactacystin (2). Elaboration of lactam 5a to the natural product was effected in 12 steps and in 11% overall yield and proceeded through an unusual spiro β-lactone intermediate (11). This compound was found to inhibit the chymotrypsin-like site of the 26S proteasome at similar levels to known inhibitor clasto-lactacystin β-lactone (omuralide)

Investigating the Initial Steps in the Biosynthesis of Cyanobacterial Sunscreen Scytonemin

Investigating the Initial Steps in the Biosynthesis of Cyanobacterial Sunscreen Scytonemi

Characterization of 1,2-Propanediol Dehydratases Reveals Distinct Mechanisms for B₁₂-Dependent and Glycyl Radical Enzymes

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of (<i>S</i>)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B₁₂-dependent propanediol dehydratase (B₁₂-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B₁₂-PD. Here we report ¹⁸O labeling experiments that suggest PD and B₁₂-PD employ distinct mechanisms. Unlike B₁₂-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1-<i>gem</i> diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions

A Prodrug Resistance Mechanism Is Involved in Colibactin Biosynthesis and Cytotoxicity

Commensal Escherichia coli residing in the human gut produce colibactin, a small-molecule genotoxin of unknown structure that has been implicated in the development of colon cancer. Colibactin biosynthesis is hypothesized to involve a prodrug resistance strategy that entails initiation of biosynthesis via construction of an N-terminal prodrug scaffold and late-stage cleavage of this structural motif during product export. Here we describe the biochemical characterization of the prodrug synthesis, elongation, and cleavage enzymes from the colibactin biosynthetic pathway. We show that nonribosomal peptide synthetases ClbN and ClbB assemble and process an <i>N</i>-acyl-d-asparagine prodrug scaffold that serves as a substrate for the periplasmic d-amino peptidase ClbP. In addition to affording information about structural features of colibactin, this work reveals the biosynthetic logic underlying the prodrug resistance strategy and suggests that cytotoxicity requires amide bond cleavage

Discovery of a Diazo-Forming Enzyme in Cremeomycin Biosynthesis

The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show that the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N–N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology as well as enable genome-based discovery of new diazo-containing metabolites

Lomaiviticin Biosynthesis Employs a New Strategy for Starter Unit Generation

Lomaiviticin biosynthesis is thought to utilize a propionyl starter unit for a type II polyketide synthase (PKS). Discovery of the lomaiviticin (<i>lom</i>) biosynthetic gene cluster suggested an unusual method for starter unit generation involving a bifunctional acyltransferase/decarboxylase (AT/DC) thus far observed only in type I PKS pathways. In vitro biochemical characterization of AT/DC Lom62 confirmed its ability to generate a propionyl-acyl carrier protein (ACP), revealing a new role for this enzymatic activity within natural product biosynthesis

Structural Analysis of Spiro β-Lactone Proteasome Inhibitors

Structural Analysis of Spiro β-Lactone Proteasome Inhibitor

Correction to Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzymes

Correction to Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzyme

Characterization of Choline Trimethylamine-Lyase Expands the Chemistry of Glycyl Radical Enzymes

The recently identified glycyl radical enzyme (GRE) homologue choline trimethylamine-lyase (CutC) participates in the anaerobic conversion of choline to trimethylamine (TMA), a widely distributed microbial metabolic transformation that occurs in the human gut and is linked to disease. The proposed biochemical function of CutC, C–N bond cleavage, represents new reactivity for the GRE family. Here we describe the in vitro characterization of CutC and its activating protein CutD. We have observed CutD-mediated formation of a glycyl radical on CutC using EPR spectroscopy and have demonstrated that activated CutC processes choline to trimethylamine and acetaldehyde. Surveys of potential alternate CutC substrates uncovered a strict specificity for choline. Homology modeling and mutagenesis experiments revealed essential CutC active site residues. Overall, this work establishes that CutC is a GRE of unique function and a molecular marker for anaerobic choline metabolism