A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Thomas J. McQuade; Abbie D. Shallop; Anita Sheoran; James E. DelProposto; Oleg V. Tsodikov; Sylvie Garneau-Tsodikova

doi:10.1016/j.ab.2008.12.014

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Thomas J. McQuade, Abbie D. Shallop, Anita Sheoran, James E. DelProposto, Oleg V. Tsodikov, Sylvie Garneau-Tsodikova

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

56 Scopus citations

Abstract

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[³²P]pyrophosphate (PP_i) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P_i) concentrations after degradation by inorganic pyrophosphatase of the PP_i released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A₄, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[³²P]PP_i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.

Original language	English
Pages (from-to)	244-250
Number of pages	7
Journal	Analytical Biochemistry
Volume	386
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2008.12.014
State	Published - Mar 15 2009

Bibliographical note

Funding Information:
This work was supported by the Life Sciences Institute and the College of Pharmacy at the University of Michigan and by a special award to S.G-T. from the Horace H. Rackham School of Graduate Studies. We thank Huan Tang Li for preliminary cloning and protein expression experiments of NcpB-A 4 . We thank Andrew King for help with protein purification. A.D.S. and Andrew King acknowledge the Undergraduate Research Opportunity Program (UROP) at the University of Michigan. Andrew King was also partially supported by the Perrigo Company and the University of Michigan Life Sciences Institute (Perrigo Undergraduate Fellowship).

Funding

This work was supported by the Life Sciences Institute and the College of Pharmacy at the University of Michigan and by a special award to S.G-T. from the Horace H. Rackham School of Graduate Studies. We thank Huan Tang Li for preliminary cloning and protein expression experiments of NcpB-A 4 . We thank Andrew King for help with protein purification. A.D.S. and Andrew King acknowledge the Undergraduate Research Opportunity Program (UROP) at the University of Michigan. Andrew King was also partially supported by the Perrigo Company and the University of Michigan Life Sciences Institute (Perrigo Undergraduate Fellowship).

Funders	Funder number
University of Michigan Life Sciences Institute
University of Michigan Hospital
Institute of Life Sciences India
Perrigo Company Charitable Foundation

Keywords

Adenylation domain
Colorimetric
Combinatorial biosynthesis
High-throughput screening
Inorganic pyrophosphatase
Malachite green
Nonradioactive
Nostocyclopeptide

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.ab.2008.12.014

Cite this

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title = "A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis",

abstract = "Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PPi) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (Pi) concentrations after degradation by inorganic pyrophosphatase of the PPi released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A4, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PPi exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.",

keywords = "Adenylation domain, Colorimetric, Combinatorial biosynthesis, High-throughput screening, Inorganic pyrophosphatase, Malachite green, Nonradioactive, Nostocyclopeptide",

author = "McQuade, {Thomas J.} and Shallop, {Abbie D.} and Anita Sheoran and DelProposto, {James E.} and Tsodikov, {Oleg V.} and Sylvie Garneau-Tsodikova",

note = "Funding Information: This work was supported by the Life Sciences Institute and the College of Pharmacy at the University of Michigan and by a special award to S.G-T. from the Horace H. Rackham School of Graduate Studies. We thank Huan Tang Li for preliminary cloning and protein expression experiments of NcpB-A 4 . We thank Andrew King for help with protein purification. A.D.S. and Andrew King acknowledge the Undergraduate Research Opportunity Program (UROP) at the University of Michigan. Andrew King was also partially supported by the Perrigo Company and the University of Michigan Life Sciences Institute (Perrigo Undergraduate Fellowship). ",

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AU - DelProposto, James E.

AU - Tsodikov, Oleg V.

AU - Garneau-Tsodikova, Sylvie

N1 - Funding Information: This work was supported by the Life Sciences Institute and the College of Pharmacy at the University of Michigan and by a special award to S.G-T. from the Horace H. Rackham School of Graduate Studies. We thank Huan Tang Li for preliminary cloning and protein expression experiments of NcpB-A 4 . We thank Andrew King for help with protein purification. A.D.S. and Andrew King acknowledge the Undergraduate Research Opportunity Program (UROP) at the University of Michigan. Andrew King was also partially supported by the Perrigo Company and the University of Michigan Life Sciences Institute (Perrigo Undergraduate Fellowship).

PY - 2009/3/15

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N2 - Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PPi) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (Pi) concentrations after degradation by inorganic pyrophosphatase of the PPi released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A4, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PPi exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.

AB - Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PPi) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (Pi) concentrations after degradation by inorganic pyrophosphatase of the PPi released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A4, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PPi exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.

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KW - Combinatorial biosynthesis

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KW - Inorganic pyrophosphatase

KW - Malachite green

KW - Nonradioactive

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JF - Analytical Biochemistry

IS - 2

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A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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