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

DOI: 10.1038/nchembio.1086 DOI Help
PMID: 23042036 PMID Help

Authors: Evelyne Deery (University of Kent) , Susanne Schroeder (University of Kent) , Andrew D. Lawrence (University of Kent) , Samantha L. Taylor (University of Kent) , Jitka Waterman (Diamond Light Source) , Keith S. Wilson , David Brown (University of Kent) , Michael A. Geeves (University of Kent) , Mark J. Howard (University of Kent) , Richard Pickersgill (Queen Mary University of London) , Martin J. Warren (University of Kent) , Arefeh Seyedarabi (Queen Mary University of London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Chemical Biology , VOL 8 (11) , PAGES 933-940

State: Published (Approved)
Published: October 2012

Open Access Open Access

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.

Journal Keywords: Escherichia; Methyltransferases; Models; Molecular; Uroporphyrins; Vitamin B 12

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography

Other Facilities: ESRF