Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

Na Pi, Caren L Meyers, Michelle Pacholec, Christopher T. Walsh, Julie A. Leary

Research output: Contribution to journalArticle

Abstract

The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

Original languageEnglish (US)
Pages (from-to)10036-10041
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number27
DOIs
StatePublished - Jul 6 2004
Externally publishedYes

Fingerprint

Novobiocin
Skeleton
Ligases
Enzymes
Anti-Bacterial Agents
Carboxyl and Carbamoyl Transferases
Methyltransferases
Transferases
Biological Products
Acids

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton. / Pi, Na; Meyers, Caren L; Pacholec, Michelle; Walsh, Christopher T.; Leary, Julie A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 27, 06.07.2004, p. 10036-10041.

Research output: Contribution to journalArticle

@article{5063e3f7f215402a9a0968ecb9e15b97,
title = "Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton",
abstract = "The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.",
author = "Na Pi and Meyers, {Caren L} and Michelle Pacholec and Walsh, {Christopher T.} and Leary, {Julie A.}",
year = "2004",
month = "7",
day = "6",
doi = "10.1073/pnas.0403526101",
language = "English (US)",
volume = "101",
pages = "10036--10041",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "27",

}

TY - JOUR

T1 - Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

AU - Pi, Na

AU - Meyers, Caren L

AU - Pacholec, Michelle

AU - Walsh, Christopher T.

AU - Leary, Julie A.

PY - 2004/7/6

Y1 - 2004/7/6

N2 - The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

AB - The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

UR - http://www.scopus.com/inward/record.url?scp=3042791444&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3042791444&partnerID=8YFLogxK

U2 - 10.1073/pnas.0403526101

DO - 10.1073/pnas.0403526101

M3 - Article

VL - 101

SP - 10036

EP - 10041

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 27

ER -