An FAD-Dependent Pyridine Nucleotide-Disulfide Oxidoreductase Is Involved in Disulfide Bond Formation in FK228 Anticancer Depsipeptide

Cheng Wang; Shane R. Wesener; Hailong Zhang; Yi Qiang Cheng

doi:10.1016/j.chembiol.2009.05.005

An FAD-Dependent Pyridine Nucleotide-Disulfide Oxidoreductase Is Involved in Disulfide Bond Formation in FK228 Anticancer Depsipeptide

Cheng Wang, Shane R. Wesener, Hailong Zhang, Yi Qiang Cheng

Research output: Contribution to journal › Article › peer-review

56 Scopus citations

Abstract

Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP⁺. We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228.

Original language	English
Pages (from-to)	585-593
Number of pages	9
Journal	Chemistry and Biology
Volume	16
Issue number	6
DOIs	https://doi.org/10.1016/j.chembiol.2009.05.005
State	Published - 26 Jun 2009

Keywords

CHEMBIO
PROTEINS

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10.1016/j.chembiol.2009.05.005

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title = "An FAD-Dependent Pyridine Nucleotide-Disulfide Oxidoreductase Is Involved in Disulfide Bond Formation in FK228 Anticancer Depsipeptide",

abstract = "Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP+. We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228.",

keywords = "CHEMBIO, PROTEINS",

author = "Cheng Wang and Wesener, {Shane R.} and Hailong Zhang and Cheng, {Yi Qiang}",

note = "Funding Information: We thank Herbert Schweizer for providing vectors pPS858, pEX18Tc, and pFLP2, and Ryan Gill for providing vector pBMTL-3. We thank Patrick Anderson for assistance with LC-MS, and Daad Saffarini for assistance with lysis of cells on a French press. We further thank Michael Thomas for critical reading of the manuscript, and we appreciate anonymous reviewers{\textquoteright} helpful comments. This work was supported by a University of Wisconsin-Milwaukee Research Growth Initiative Award and by a US Department of Defense Breast Cancer Research Program Idea Award W81XWH-08-1-0673. ",

year = "2009",

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doi = "10.1016/j.chembiol.2009.05.005",

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AU - Wang, Cheng

AU - Wesener, Shane R.

AU - Zhang, Hailong

AU - Cheng, Yi Qiang

N1 - Funding Information: We thank Herbert Schweizer for providing vectors pPS858, pEX18Tc, and pFLP2, and Ryan Gill for providing vector pBMTL-3. We thank Patrick Anderson for assistance with LC-MS, and Daad Saffarini for assistance with lysis of cells on a French press. We further thank Michael Thomas for critical reading of the manuscript, and we appreciate anonymous reviewers’ helpful comments. This work was supported by a University of Wisconsin-Milwaukee Research Growth Initiative Award and by a US Department of Defense Breast Cancer Research Program Idea Award W81XWH-08-1-0673.

PY - 2009/6/26

Y1 - 2009/6/26

N2 - Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP+. We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228.

AB - Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP+. We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228.

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KW - PROTEINS

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An FAD-Dependent Pyridine Nucleotide-Disulfide Oxidoreductase Is Involved in Disulfide Bond Formation in FK228 Anticancer Depsipeptide

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