Abstract
The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B12) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.
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Acknowledgements
This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/E024203 and BB/I013334) and the Wellcome Trust (091163/Z/10/Z). We thank M. Rowe for additional NMR technical support. Diffraction data were collected at the European Synchrotron Radiation Facility, Grenoble, France (for CobE) and the Diamond Light Source, Oxfordshire, UK (for CobL and CobH). We thank C. Roessner (Texas A&M University) for a clone of the P. denitrificans cobG.
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E.D. designed and performed most of the experiments and analysis with support from A.D.L. and S.S.; A.D.L. performed MS analysis. S.L.T. performed all NMR data acquisition, which was analyzed with M.J.H. S.S., A.S. and R.W.P. contributed the CobLC and CobH–HBA structures and CobL and CobE–HBA models. J.W. and K.S.W. contributed the CobE structure. D.B. and S.S. determined the CobH–5-desmethyl-HBA structure. M.A.G. provided insight into substrate channeling. M.J.W. directed all aspects of the project. E.D. and M.J.W. wrote the manuscript.
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Deery, E., Schroeder, S., Lawrence, A. et al. An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nat Chem Biol 8, 933–940 (2012). https://doi.org/10.1038/nchembio.1086
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DOI: https://doi.org/10.1038/nchembio.1086
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