Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC

“…It is perhaps surprising that Fe1 is assigned to be Fe­(III), as simple considerations of charge interactions suggest that an Fe­(III) ion would likely favor coordination by more negatively charged carboxyl ligands. Importantly, a similar approach could be applied to other MNIOs, which also have, or are predicted to have, one Fe ion coordinated by a single histidine and the other coordinated by two histidines. , …”

“…Importantly, a similar approach could be applied to other MNIOs, which also have, or are predicted to have, one Fe ion coordinated by a single histidine and the other coordinated by two histidines. 19,21…”

“…The first of these subfamily members to be characterized is the MbnBC enzyme complex, which catalyzes the key step , in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, post-translationally modified peptide (RiPP) natural products that bind copper with high affinity. ,, Mbns, produced by some methane-oxidizing (methanotrophic) bacteria under copper starvation conditions, show promise as therapeutics for Wilson disease, a genetic disorder of copper metabolism. − However, further development and engineering of Mbn as a therapeutic requires detailed knowledge of its biosynthetic pathway. MbnBC converts cysteine residues in the Mbn precursor peptide MbnA to oxazolone rings with adjacent thioamide groups, a four-electron oxidation reaction (Figure a). , Other recently characterized DUF692 proteins also function in natural product biosynthesis, including the 3-thiaglutamate biosynthetic system, TgIHI, which generates 3-thiaglutamate − and ChrHI, which is involved in biosynthesis of the natural product chryseobasin . These combined studies have led to a renaming of the DUF692 family as multinuclear nonheme iron-dependent oxidative enzymes (MNIOs) …”

Unusual cysteine modifications in natural product biosynthesis

“…Very recently, the X-ray crystal structures of TglHI complexed with and without iron ions were reported. 75 TglH adopts a TIM barrel structure with β-sheets wrapped around helices, forming a stable complex with TglI, in a similar manner to MbnBC. 72,76 These structures revealed the formation of a catalytic pocket between TglH and TglI, and two iron–iron binding sites in the pocket and a third auxiliary iron binding site were identified.…”

Genome Mining for New Enzyme Chemistry

“…All characterized MNIOs (MbnB, TglH, ChrH) are iron-dependent. The crystal structures of MbnBC and TglHI revealed two to three irons in the active sites of MbnB and TglH. − The crystal structure of MbnBC bound to MbnA, the AlphaFold model of TglHI with the precursor TglA, and biochemical assays demonstrated that MbnC (WP_003614727.1) and TglI engage their cognate precursor peptides. , In the MbnABC structure, one Cys residue of MbnA is ligated to an iron. Comprehensive activity assays of MbnBC under varying redox conditions, complemented by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy, suggested MbnB’s reliance on a mixed iron oxidation state [Fe­(II) and Fe­(III)] for functionality .…”