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Abstract Oxalate decarboxylase fromBacillus subtilisis a binuclear Mn‐dependent acid stress response enzyme that converts the mono‐anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π‐stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long‐range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5‐hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5‐hydroxytryptophan with its hydroxyl proton removed. 5‐Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N‐terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long‐range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5‐hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.
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Discovery and Functional Characterization of SnFDHal, an Efficient Tryptophan 5-Halogenase from Streptomyces noursei
Abstract Flavin-dependent halogenases (FDHs) are a class of enzymes renowned for their regioselective ability to precisely insert halogen atoms into small aromatic compounds. Halogen incorporation can enhance the physicochemical and biological properties of molecules, making them valuable for agrochemical and pharmaceutical applications. Through bioinformatic mining of bacterial genomes, we discovered and functionally characterized SnFDHal, an efficient tryptophan 5-halogenase fromStreptomyces nourseiNRRL B-1714. This halogenase operates across a broad pH range and exhibits a melting temperature of 46.7 °C at both pH 6 and pH 8, which is comparable to thermophilic halogenases such as Th-Hal and BorH, and notably higher than those of mesophilic counterparts. Steady-state kinetic analysis revealed that SnFDHal displays superior catalytic efficiency for the chlorination of L-tryptophan compared to other FDHs reported to date. Structural modeling of its active site suggests that a conserved bulky phenylalanine residue (F49) promotes halogenation at the C-5 position of L-tryptophan, consistent with experimental findings. The combination of high catalytic efficiency and thermostability positions SnFDHal as a promising biocatalyst for applications in agrochemical and pharmaceutical industries.
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- Award ID(s):
- 2044558
- PAR ID:
- 10646627
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Applied Biochemistry and Biotechnology
- ISSN:
- 0273-2289
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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