An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis

Deery, Evelyne, Schroeder, Susanne, Lawrence, Andrew D., Taylor, Samantha L., Seyedarabi, Arefeh, Waterman, Jitka, Wilson, Keith S., Brown, David, Geeves, Michael A., Howard, Mark J., Pickersgill, Richard W. and Warren, Martin J. ORCID: https://orcid.org/0000-0002-6028-6456 (2012) An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nature Chemical Biology, 8 (11). pp. 933-940. ISSN 1552-4450

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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 B 12) 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.

Item Type: Article
Additional Information: Funding Information: 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.
Uncontrolled Keywords: molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1312
Faculty \ School: Faculty of Science
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Depositing User: LivePure Connector
Date Deposited: 20 Sep 2022 15:30
Last Modified: 21 Sep 2022 00:23
URI: https://ueaeprints.uea.ac.uk/id/eprint/88508
DOI: 10.1038/nchembio.1086

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