The enzymology of oxazolone and thioamide synthesis in methanobactin

Chou, Jonathan Chiu-Chun; Stafford, Veronica E; Kenney, Grace E.; Dassama, Laura M. K.

doi:10.1016/bs.mie.2021.04.008

Cited by 3 publications

(4 citation statements)

References 33 publications

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“…The TglHI complex is reminiscent of the recently reported holoenzyme, MbnBC, 32 , 33 which generates an oxazolone and adjacent thioamide during the biosynthesis of methanobactin (Mbn, Figure S1C ). 27 , 34 MbnB is an iron-dependent enzyme from the DUF692 protein family, and MbnC acts as a recognition subunit for MbnA recruitment to the MbnBC holoenzyme. TglH shares only 16% amino acid sequence identity with MbnB ( Figure S1D ), and the TglH-catalyzed reaction is very different from that catalyzed by MbnB.…”

Section: Introductionmentioning

confidence: 99%

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Zheng,

Xu,

et al. 2023

Structure

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Section: Introductionmentioning

confidence: 99%

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Zheng,

Xu,

et al. 2023

Structure

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“…This complex set of reactions is catalyzed by the proteins TglH and TglI, which are insoluble separately but form a soluble TglHI complex upon coexpression of His 6 -TglH with TglI in Escherichia coli . TglH, the putative catalytic subunit, is a member of the DUF692 protein family (Pfam: PF05114), which also includes the nonheme iron oxidase MbnB, the catalytic subunit of the MbnBC complex that generates the oxazolone and thioamide moieties of methanobactin. − The use of nonheme iron oxygenases and other radical-utilizing enzymes to form and break C–S bonds has been observed during the biosynthesis of a variety of secondary metabolites, such as sactipeptides and other cyclic thioether-containing RiPPs, , ergothioneine, and quinohemoprotein amine dehydrogenase . The reactions catalyzed by TglHI and MbnBC are unique, however, and their mechanisms are incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycine Synthase TglHI during 3-Thiaglutamate Biosynthesis

McLaughlin

Donk

2022

ACS Chem. Biol.

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3-Thiaglutamate is a recently identified amino acid analog originating from cysteine. During its biosynthesis, cysteinyl-tRNA is first enzymatically appended to the C-terminus of TglA, a 50-residue ribosomally translated peptide scaffold. After hydrolytic removal of the tRNA, this cysteine residue undergoes modification on the scaffold before eventual proteolysis of the nascent 3-thiaglutamyl residue to release 3-thiaglutamate and regenerate TglA. One of the modifications of TglACys requires a complex of two polypeptides, TglH and TglI, which uses nonheme iron and O 2 to catalyze the removal of the peptidyl-cysteine β-methylene group, oxidation of this Cβ atom to formate, and reattachment of the thiol group to the α carbon. Herein, we use in vitro transcription-coupled translation and expressed protein ligation to characterize the role of the TglA scaffold in TglHI recognition and determine the specificity of TglHI with respect to the C-terminal residues of its substrate TglACys. The results of these experiments establish a synthetically accessible TglACys fragment sufficient for modification by TglHI and identify the l -selenocysteine analog of TglACys, TglASec, as an inhibitor of TglHI. These insights as well as a predicted structure and native mass spectrometry data set the stage for deeper mechanistic investigation of the complex TglHI-catalyzed reaction.

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“…The gene encoding the MB precursor peptide, mbnA ( 5 , 10 ), is found in a gene cluster that contains both genes of known function, such as mbnB ( 5 , 11 ), mbnN ( 9 ), and mbnT ( 12 ), as well as unannotated genes, such as mbnC ( 5 , 10 , 11 , 13 , 14 ). MbnB is a member of TIM barrel family as well as the DUF692 family of diiron enzymes ( 11 , 14 ). In heterologous expression studies in Escherichia coli , MbnBC was shown to catalyze a dioxygen-dependent four-electron oxidation of Pro-Cys in MbnA ( 11 , 14 , 15 ).…”

Section: Introductionmentioning

confidence: 99%

“…MbnB is a member of TIM barrel family as well as the DUF692 family of diiron enzymes ( 11 , 14 ). In heterologous expression studies in Escherichia coli , MbnBC was shown to catalyze a dioxygen-dependent four-electron oxidation of Pro-Cys in MbnA ( 11 , 14 , 15 ). The roles of MbnB and MbnC could not be separately determined as attempts to separately purify these gene products in E. coli failed ( 11 ).…”

Section: Introductionmentioning

confidence: 99%

MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b

Dershwitz

Roche

et al. 2022

Appl Environ Microbiol

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Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b, mbnA , which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC , is found in all MB operons, and in conjunction with mbnB , is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC , a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the Δ mbn C mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from Δ mbn C was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals, nanoparticle formation, as well as for the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for post-translational modification(s) of the two oxazolone groups are not identical.

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The enzymology of oxazolone and thioamide synthesis in methanobactin

Cited by 3 publications

References 33 publications

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycine Synthase TglHI during 3-Thiaglutamate Biosynthesis

MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b

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