Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion1. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics2. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes3, providing information regarding the general mechanism of enzymatic oxygen activation.
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We are grateful to A. Heroux and the staff at the Brookhaven National Synchrotron Light Source PXRR for assistance with X-ray data collection and processing. We are grateful to C. C. Tseng, R. S. Klausen and C. T. Walsh for discussions and experimental assistance. We thank P. D. Fortin and A. R. Howard-Jones for assistance in oxygen-binding measurements, Z. Dauter for advice on X-ray data analysis, and members of the Bruner research group and Boston College Chemistry Department for helpful discussions and comments on the manuscript. This work was supported with funds from Boston College and the Damon Runyon Cancer Research Foundation.
Author Contributions P.F.W. crystallized and solved the structure of apo-DpgC. E.N.F. crystallized the DpgC/inhibitor co-complex and with P.F.W. solved the structure. E.N.F constructed and with P.F.W. performed biochemical assays on mutant DpgC enzymes. Y.L. synthesized and biochemically evaluated the inhibitor, DPA-NH-CoA. S.D.B. wrote the manuscript and all authors discussed the results and commented on the manuscript.
The atomic coordinates for the DpgC/inhibitor complex structure have been deposited in the Protein Data Bank with accession code 2NP9.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
This file contains Supplementary Figures S1–S4, Supplementary Table 1 and Supplementary Methods. The Supplementary Figures illustrate additional electron density maps, plots of kinetic data characterizing the synthetic inhibitor and mutant enzymes, experimental data for determination of multimeric state and a plot of data from O2 kinetic binding experiments. A Supplementary Table is included with crystallographic statistics. The Supplementary Methods section describes the cloning and purification of DpgC, enzyme kinetic assays, site-directed mutagenesis, molecular weight determination and synthesis of the inhibitor DPA-NH-CoA. (PDF 1123 kb)
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Widboom, P., Fielding, E., Liu, Y. et al. Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis. Nature 447, 342–345 (2007). https://doi.org/10.1038/nature05702
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