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

Abstract

The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B12) pathway in E. 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 catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis

“…values of 1.6, 1.7, and 1.9 Å, respectively, for all C α atoms (Figure 3b). Recently, the crystal structure of the C-terminal region of CobL (CobL c ) from Rhodobacter capsulatus was reported [24]. The enzyme CobL, which uses precorrin-6B as a substrate, promotes the decarboxylation of the acetic acid side chain at C12, and the methylation at the C5 & C15 meso positions, to produce precorrin-8X.…”

The Uses and Future Prospects of Metabolomics and Targeted Metabolite Profiling in Cell Factory Development

“…Deery et al introduced a new metabolomics-driven approach for mapping metabolic pathways in cases in which the genes are known but the chemical structures of the intermediates have not been elucidated, such as the cobalamin biosynthetic pathway in bacteria. 38 Their approach, named enzyme-trap, is based on protein pull-down followed by liquid chromatography (LC)/MS and nuclear magnetic resonance (NMR) based identification of the metabolites that are transiently bound to the catalytic enzymes.…”

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B 12 )

“…The cobalt-late or aerobic pathway is the better characterized, and all of the intermediates and enzymes responsible for the de novo construction of the corrin component have now been determined (3,4). In contrast, the cobalt-early or anaerobic pathway is poorly delineated.…”

“…For the rest of the pathway, the intermediates and reactions have yet to be isolated. Indeed, the enzymes CbiJ, CbiE, CbiT, and CbiC share reasonable homology (∼20-40% amino acid identity) to enzymes from the aerobic pathway that have been characterized and shown to convert precorrin-6 to hydrogenobyrinic acid (3,4). A summary of the aerobic and anaerobic pathways is shown in Fig.…”