期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：27
页码：10036-10041
DOI：10.1073/pnas.0403526101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
