title: An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.
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.
authors.affiliation: School of Biosciences, University of Kent, Canterbury, Kent, UK.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Deery
authors.forename: Susanne
authors.initials: S
authors.lastname: Schroeder
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Lawrence
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Taylor
authors.forename: Arefeh
authors.initials: A
authors.lastname: Seyedarabi
authors.forename: Jitka
authors.initials: J
authors.lastname: Waterman
authors.forename: Keith S
authors.initials: KS
authors.lastname: Wilson
authors.forename: David
authors.initials: D
authors.lastname: Brown
authors.forename: Michael A
authors.initials: MA
authors.lastname: Geeves
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Howard
authors.forename: Richard W
authors.initials: RW
authors.lastname: Pickersgill
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
mesh_terms: D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D014578:Uroporphyrins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014805:Vitamin B 12 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D014578:Uroporphyrins; C107384:precorrin 4; C072476:hydrogenobyrinic acid; D008780:Methyltransferases; D014805:Vitamin B 12