An Alternative Active Site Architecture for O₂ Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum

Abstract

Sulfoxide synthases are non-heme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-a-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the g-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five sub-types, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.

Selenocysteine as a Substrate, an Inhibitor and a Mechanistic Probe for Bacterial and Fungal Iron‐Dependent Sulfoxide Synthases

“…The same compound has been implicated as an intermediate in the biosynthesis of 2 by S. pombe . In the presence of 0.5 m m SeCys and 0.5 m m TMH, Cth EgtB produces 4 at a rate of (1.2±0.2)×10 −3 s −1 (Figure S3, Supporting Information) which is roughly 200‐fold slower than formation of sulfoxide 3 under the same conditions . We did not observe selenoxide 5 , which is not surprising because selenoxides are rapidly reduced in the presence of selenols, ascorbate or TCEP…”

“…We have previously described the crystal structure of Cth EgtB and shown that this type of bacterial sulfoxide synthase accepts TMH and Cys as substrates . At neutral pH and at room temperature, Cth EgtB produces sulfoxide 3 as the main product (80 %) and cysteine dioxide as a minor product ( 6 , 20 %, Figure ).…”

“…At neutral pH and at room temperature, Cth EgtB produces sulfoxide 3 as the main product (80 %) and cysteine dioxide as a minor product ( 6 , 20 %, Figure ). Under steady‐state conditions, consumption of Cys is characterized by a k cat of 0.14 s −1 and apparent K m values of 27 μ m for Cys and 65 μ m for TMH . Quantification of SeCys consumption by the same assay was complicated by the considerable reactivity of selenols towards O 2 and reducing agents.…”

“… Cth EgtB catalyzes the production of sulfoxide 3 which is converted to ergothioneine ( 1 ) by a PLP‐dependent β‐lyase . In presence of SeCys Cth EgtB can catalyze the formation of the selenoether 4 .…”

Convergent Evolution of Fungal Cysteine Dioxygenases

“…The evolutionary emergence of type II CDOs was probably facilitated by the fact that a single point mutation in sulfoxide synthases suffice to elicit efficient thiol dioxygenase activity. [21,22] As DsqCDO retained the ability to bind TMH, we wondered as to whether mutating Phe442 to Tyr would restore sulfoxide synthase activity. To address this question, we examined the corresponding variant (DsqCDO F442Y ) by the same methodology as described above.…”