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

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Author(s)

  • Evelyne Deery
  • Susanne Schroeder
  • Andrew D. Lawrence
  • Samantha L. Taylor
  • Arefeh Seyedarabi
  • Jitka Waterman
  • Keith S. Wilson
  • David Brown
  • Michael A. Geeves
  • Mark J. Howard
  • Richard W. Pickersgill
  • Martin J. Warren

Department/unit(s)

Publication details

JournalNATURE CHEMICAL BIOLOGY
DatePublished - Nov 2012
Issue number11
Volume8
Number of pages8
Pages (from-to)933-940
Original languageEnglish

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.

    Research areas

  • RHODOBACTER-CAPSULATUS, PSEUDOMONAS-DENITRIFICANS, PURIFICATION, HYDROGENOBYRINIC ACID, MAXIMUM-LIKELIHOOD, RING CONTRACTION, PRECORRIN-8X, GENE-PRODUCT, VITAMIN-B12 BIOSYNTHESIS, SUBSTRATE

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