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Biochemistry. 2014 Jan 21;53(2):413-22. doi: 10.1021/bi401529y. Epub 2014 Jan 7.

Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases.

Author information

1
Department of Chemistry, ‡Institute for Genomic Biology, and §Department of Microbiology; University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

Abstract

Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown neither to alter complex formation with the cyclodehydratase nor the reduction potential. Taken together with the known crystal structure of a homologue, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis.

PMID:
24364559
PMCID:
PMC3928670
DOI:
10.1021/bi401529y
[Indexed for MEDLINE]
Free PMC Article

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