Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis: A [4Fe-4S] CLUSTER AND THE CATALYSIS OF A RADICAL S-ADENOSYLMETHIONINE ENZYME

Qi Zhang,Dandan Chen,Jun Lin,Rijing Liao,Wei Tong,Zhinan Xu,Wen Liu

doi:10.1074/jbc.m111.224832

Abstract

The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis. EPR and UV-visible absorbance spectroscopic analyses demonstrated the interaction between L-Trp and the [4Fe-4S] cluster of NocL, leading to the assumption of nonspecific interaction of [4Fe-4S] cluster with other nucleophiles via the unique Fe site. This notion is supported by the finding of the heterogeneity in the [4Fe-4S] cluster of NocL in the absence of AdoMet, which was revealed by the EPR study at very low temperature. Furthermore, a free radical was observed by EPR during the catalysis, which is in good agreement with the hypothesis of a glycyl radical intermediate. Combined with the mutational analysis, these studies provide new insights into the function of the [4Fe-4S] cluster of radical AdoMet enzymes as well as the mechanism of the radical-mediated complex carbon chain rearrangement catalyzed by MIA synthase.

Highlights

Fe-S clusters are ubiquitous and ancient prosthetic groups that are essential for a wide range of biological processes [1,2,3,4]
We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism
We report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis

Summary

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. Since the discovery of the first member, lysine 2,3-aminomutase, in 1970 [11], the radical AdoMet superfamily is believed to comprise of thousands of proteins that participate in numerous biochemical processes in animals, plants, and microorganisms These enzymes contain a motif of three cysteine residues (usually as CXXXCXXC) that nucleate a [4Fe-4S] cluster for binding of AdoMet via a unique Fe (Feu) site uncoordinated to the cysteine residue. We recently demonstrated that NosL, a radical AdoMet enzyme involved in biosynthesis of the thiopeptide antibiotic nosiheptide (NOS) (Fig. 1A), catalyzes the rearrangement of L-Trp to afford 3-methyl-2-indolic acid (MIA) [15, 16]. This indicates that NosL homologs might be involved in the biosynthetic pathways of all the e-series

Characterization of NocL as a Radical AdoMet MIA Synthase

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUSION