Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan

Abstract

Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3-methyl-2-indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan CaÀCb bond and recombination of the amino-acid-derived -COOH fragment at the indole ring. Reported herein is the 1.8 resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for H abstraction by the SAM-derived 5'-deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Ca À Cb bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a CNH tryptophanyl radical. A structure-based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent CNH radical operates in the latter enzymes.Scheme 1. Reaction catalyzed by NosL. The carboxylate fragment in MIA (depicted in bold) originates directly from tryptophan. [4]

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

“…12 To test this prediction in a biochemical assay, the NosL reaction was run using a tryptophan analog 14 in which the indole NH is replaced by sulfur. This reaction generated 15 and 16 as products consistent with hydrogen atom abstraction from the amino group (see Figures S4-S5).…”

The Promiscuous Activity of the Radical SAM Enzyme NosL toward Two Unnatural Substrates

“…In the NosL crystal structure in complex with tryptamine (PDB ID: 6HTM), [ 34 ] none of the enzyme active site residues has an apparent move compared to those in the L ‐Trp‐bound structure (PDB ID: 4R34). [ 26 ] This observation suggests that tryptamine is bound in a similar microenvironment as that of L ‐Trp (Figure S1), questioning whether the tryptamine C1—C2 bond (analogous to the L ‐Trp Cα—Cβ bond) could be cleaved. We ran NosL reaction by incubating tryptamine with the reconstituted NosL, SAM, and dithionite.…”

“…[ 21‐25 ] In the case of NosL, the dAdo radical abstracts a hydrogen atom from the L ‐Trp amino group to produce a nitrogen‐ centered tryptophanyl radical 3 . [ 26‐28 ] The Cα—COO – bond is then cleaved to release a carboxyl radical •CO 2 – 5 , which adds to the indole C2 of indole‐3‐ethanimine ( 4 ) to produce 6 , a stable radical that can be well characterized by electron paramagnetic resonance (EPR) spectroscopy. [ 11 ] 6 is then converted to a cyanide and MIA by an as‐yet unknown mechanism.…”

Identification of truncated form of NosP as a transcription factor to regulate the biosynthesis of nosiheptide

“…In 2009, a 16-gene cluster for the biosynthesis of NOS, a representative thiopeptide antibiotic, was reported (24). Since then, the functions of a number of genes have been gradually elucidated to partially complete the biosynthetic pathway of NOS (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). However, we still lack of any knowledge about the pathway-specific regulation, although the nosP gene was predicted to encode a SARP family regulatory protein.…”

“…With the discovery of the nos gene cluster in 2009, the biosynthesis of NOS was revealed to be a ribosomally synthesized and posttranslationally modified peptide pathway, and the necessity of several genes, including nosM, nosH, nosL, and nosN, for NOS maturation was verified in vivo or in vitro (24). Subsequently, the roles of many genes in the nos cluster, such as nosA (25-28), nosB (27), nosC (27), nosI (29), nosJ (29), nosK (29), nosL (30,31), nosM (26,(32)(33)(34), and nosN (35), have been elucidated, which lays a foundation for a better understanding of the biosynthetic machinery for NOS. Within the nos gene cluster, the gene nosP was proposed as a regulatory gene based on sequence homolog.…”