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 ionization͞MS 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 (NovL͞NovM) and three-enzyme (NovL͞NovM͞NovP) 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.

The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

“…; 24 Studies with NovP and its orthologue CloP, have shown that both enzymes are capable of methylating unnatural substrates 2526 The crystal structure of NovP presented here may provide explanations for these phenomena and, moreover, identify ways of manipulating the enzyme towards enhancing its in vitro activity and substrate promiscuity.…”

Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose–ketose isomers using tandem mass spectrometry

“…However, the total assay time, over 50 min for each run, is quite long for a kinetic assay. Our laboratory has been working on developing different mass spectrometry based assays to directly study steady state kinetics and mechanisms of various enzyme systems [26][27][28][29][30][31][32][33]. Using single ion monitoring to follow the formation of products during the reaction process, the kinetic parameters of a sulfurtransferase [27,33] and a phosphotransferase [29] have been successfully measured.…”

“…Using single ion monitoring to follow the formation of products during the reaction process, the kinetic parameters of a sulfurtransferase [27,33] and a phosphotransferase [29] have been successfully measured. A similar methodology was applied to follow the process of a multi-enzyme system, in which reaction intermediates and products at different stages were monitored simultaneously based on their masses [32].…”

Structure and Mechanism of the Rebeccamycin Sugar 4′-O-Methyltransferase RebM

“…1B) (17). RebM displays some subtle distinctions from the other sugar O-MTases studied in vitro including oleandomycin OleY (19), tylosin TylE and TylF (20,21), mycinamicin MycF (22), coumermycin CouP (23), and novobiocin NovP (24). In contrast to typical multimeric methyltransferases (19 -21, 25-27), RebM functions as a monomer.…”