Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product

Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine during the biosynthesis of the metallo-cluster of the [Fe–Fe] hydrogenase

Tryptophan Lyase (NosL): A Cornucopia of 5′-Deoxyadenosyl Radical Mediated Transformations

Tryptophan lyase (NosL) is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that catalyzes the formation of 3-methyl-2-indolic acid from l-tryptophan. In this paper, we demonstrate that the 5′-deoxyadenosyl radical is considerably more versatile in its chemistry than previously anticipated: hydrogen atom abstraction from N_α-cyclopropyltryptophan occurs at Cα rather than the amino group with NosL Y90A and replacing the substrate amine with a ketone or an alkene changes the chemistry from hydrogen atom abstraction to double bond addition. In addition, the 5′-deoxyadenosyl radical can add to the [4Fe−4S] cluster and dithionite can be used to trap radicals at the active site

Menaquinone Biosynthesis: Formation of Aminofutalosine Requires a Unique Radical SAM Enzyme

Menaquinone (MK, vitamin K₂) is a lipid-soluble molecule that participates in the bacterial electron transport chain. In mammalian cells, MK functions as an essential vitamin for the activation of various proteins involved in blood clotting and bone metabolism. Recently, a new pathway for the biosynthesis of this cofactor was discovered in <i>Streptomyces coelicolor</i> A3(2) in which chorismate is converted to aminofutalosine in a reaction catalyzed by MqnA and an unidentified enzyme. Here, we reconstitute the biosynthesis of aminofutalosine and demonstrate that the missing enzyme (aminofutalosine synthase, MqnE) is a radical SAM enzyme that catalyzes the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl)­oxy]­benzoic acid. This is a new reaction type in the radical SAM superfamily

In vitro reconstitution of the radical S-adenosylmethionine enzyme MqnC involved in the biosynthesis of futalosine-derived menaquinone

The radical S-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the in vitro reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical. © 2013 American Chemical Society

Aminofutalosine Synthase: Evidence for Captodative and Aryl Radical Intermediates Using β‑Scission and S_RN1 Trapping Reactions

Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the menaquinone biosynthetic pathway. In this communication, we propose a novel mechanism for this reaction involving the addition of the adenosyl radical to the substrate double bond to form a captodative radical followed by rearrangement and decarboxylation to form an aryl radical anion which is then oxidized by the [4Fe–4S]⁺² cluster. Consistent with this proposal, we describe the trapping of the captodative radical and the aryl radical anion using radical triggered C–Br fragmentation reactions. We also describe the trapping of the captodative radical by replacing the vinylic carboxylic acid with an amide

<i>In Vitro</i> Reconstitution of the Radical <i>S</i>‑Adenosylmethionine Enzyme MqnC Involved in the Biosynthesis of Futalosine-Derived Menaquinone

The radical <i>S</i>-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the <i>in vitro</i> reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical

Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: A treasure trove of complex organic radical rearrangement reactions

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry

Polyketide Ring Expansion Mediated by a Thioesterase, Chain Elongation and Cyclization Domain, in Azinomycin Biosynthesis: Characterization of AziB and AziG

The azinomycins are a family of potent antitumor agents with the ability to form interstrand cross-links with DNA. This study reports on the unusual biosynthetic formation of the 5-methyl naphthoate moiety, which is essential for effective DNA association. While sequence analysis predicts that the polyketide synthase (AziB) catalyzes the formation of this naphthoate, 2-methylbenzoic acid, a truncated single-ring product, is formed instead. We demonstrate that the thioesterase (AziG) acts as a chain elongation and cyclization (CEC) domain and is required for the additional two rounds of chain extension to form the expected product