Single-Step Replacement of an Unreactive C–H Bond by a C–S Bond Using Polysulfide as the Direct Sulfur Source in the Anaerobic Ergothioneine Biosynthesis

Abstract

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosyntheses, non-heme iron enzymes incorporate a sulfoxide into an sp 2 C−H bond from trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green-sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB), directly replaces this unreactive hercynine C−H bond with a C−S bond. Herein, we demonstrate that polysulfide (HSS n SR) is the direct sulfur source in EanB catalysis. After identifying EanB's substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that the protonation of N π of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp 2 C−H bond in this trans-sulfuration reaction.

In Vitro Production of Ergothioneine Isotopologues

“…Upon further analysis we realized that soluble polysulfides generated from partially oxidized hydrogen disulfide can also serve as the primary sulfur donor [16b, 19] . This finding has been confirmed recently by an independent study [20] …”

“…[16b, 19] This finding has been confirmed recently by an independent study. [20] Measuring the rates of ergothioneine production at concentrations of Na 2 S ranging from 1 to 50 mM showed that the enzyme reaches saturation at 5 mM (entries 2-6, Table 1). At this concentration EanB catalyzes TMH sulfurization at a rate of 1 min À1 , which is twofold faster that previously determined as maximal velocity under single turnover conditions (entry 1).…”

OvoAMthtfromMethyloversatilis thermotoleransovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities

“…5,17–19 One ovothiol biosynthetic pathway has also been biochemically characterized (Scheme 1). 9,10 In these pathways, the key step is the activation of imidazole sp 2 C–H bonds and replacing them with a C–S bond (catalyzed by EgtB, 12 Egt1, 14 EanB, 17,18 and OvoA, 9,20 Scheme 1). In the aerobic ergothioneine biosynthetic pathways from Mycobacterium smegmatis and Neurospora crassa , a non-heme iron enzyme (EgtB 12 or Egt1 (ref.…”

“…In the last decade, one anaerobic and two aerobic 12,14,15 ergothioneine biosynthetic pathways have been discovered (Scheme 1). 5,[17][18][19] One ovothiol biosynthetic pathway has also been biochemically characterized (Scheme 1). 9,10 In these pathways, the key step is the activation of imidazole sp 2 C-H bonds and replacing them with a C-S bond (catalyzed by EgtB, 12 Egt1, 14 EanB, 17,18 and OvoA, 9,20 Scheme 1).…”

Metabolic Adaptations to Marine Environments: Molecular Diversity and Evolution of Ovothiol Biosynthesis in Bacteria

“…On the other hand, the first appearance of oxygen in the primordial atmosphere could have represented a selective pressure for the evolution of oxygen-dependent sulfoxidation. Interestingly, an alternative anaerobic pathway for the biosynthesis of the ergothioneine has been previously described in the green-sulfur bacterium, Chlorobium limicola ( Leisinger et al 2019 ), in which a rhodanese domain containing protein participate to the formation of a C − S bond, using polysulfide as the direct sulfur source ( Cheng et al 2020 ). Among the two strictly anaerobic bacteria displaying OvoA-like sequences, S. hongkongensis is involved in marine sedimentary sulfur oxidation and denitrification: it grew chemolithoautotrophically using thiosulfate, sulfide, or hydrogen as electron donor and nitrate as the electron acceptor under anoxic conditions ( Chen and Dong 2005 ).…”