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

Proceedings of the National Academy of Sciences of the United States of America
Na PiJulie A Leary

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 p...Continue Reading

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Citations

Jul 12, 2005·Analytical and Bioanalytical Chemistry·André Liesener, Uwe Karst
Feb 20, 1993·Biochimica Et Biophysica Acta·M NishimotoK Okuda
Jun 17, 2011·Biochemistry·Mahendra S SandbhorChristopher W Cairo
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Sep 26, 2009·Natural Product Reports·Lutz Heide
Jan 27, 2005·Journal of Mass Spectrometry : JMS

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