Cysteine Dealkylation in Bacillus subtilis by a Novel Flavin-Dependent Monooxygenase.

In this paper, we describe the biochemical reconstitution of a cysteine salvage pathway and the biochemical characterization of each of the five enzymes involved. The salvage begins with amine acetylation of S-alkylcysteine, followed by thioether oxidation. The C-S bond of the resulting sulfoxide is cleaved using a new flavoenzyme catalytic motif to give N-acetylcysteine sulfenic acid. This is then reduced to the thiol and deacetylated to complete the salvage pathway. We propose that this pathway is important in the catabolism of alkylated cysteine generated by proteolysis of alkylated glutathione formed in the detoxification of a wide range of electrophiles.

Mechanistic Studies on the Radical SAM Enzyme Tryptophan Lyase (NosL).

Tryptophan lyase (NosL) is a radical SAM enzyme that catalyzes the formation of 3-methyl-2-indolic acid from l-tryptophan in the biosynthesis of the antibiotic nosiheptide. NosL is the newest addition to the radical SAM-dependent aromatic amino acid lyase subfamily which includes ThiH, HydG, and CofH. The recently solved crystal structure of NosL challenged the previously accepted mechanistic hypothesis and spurred a renewed interest in investigating the reaction. This led to a series of studies that unraveled several fascinating aspects of the fragmentation-recombination reaction. This chapter describes the various methodologies used for the overexpression of NosL, its purification, in vitro reconstitution, preparation of isotopically labeled substrates, and chemoenzymatic synthesis of substrate analogs. The methods described here can be used to further investigate other aromatic amino acid lyases as well as reactivity of fleeting radicals in enzymology.

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 S-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 Nα-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.

Tryptophan Lyase (NosL): Mechanistic Insights from Substrate Analogues and Mutagenesis.

NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cß bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first ß-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the ß-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom.