Functional and structural characterization of an ECF-type ABC transporter for vitamin B12.

Joana A Santos,Stephan Rempel,Jan-Willem De Gier,Josy Ter Beek,Sandra Tm Mous,Dirk J Slotboom,Albert Guskov,Cristiane T Pereira

doi:10.7554/elife.35828

Joana A Santos, Stephan Rempel + Show 6 more

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https://doi.org/10.7554/elife.35828

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Abstract

Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.

Highlights

Vitamin B12 or cobalamin (Cbl) is regarded as the largest and most complex biological ‘small molecule’
Because btuF and btuC are deleted in E. coli DFEC, endogenous Cbl-uptake mediated by BtuCDF is abolished (Cadieux et al, 2002), and expression of an active Cbl transporter is required to synthesize methionine
In L. delbrueckii, which was shown to be auxotrophic for vitamin B12 (Kusaka and Kitahara, 1962), CbrT occurs within the nrdJ–cbrS–cbrT–pduO gene cluster

Summary

Introduction

Vitamin B12 or cobalamin (Cbl) is regarded as the largest and most complex biological ‘small molecule’. At the a-axial position, the cobalt ion is coordinated by the 5,6-dimethylbenzimidazole (DMBI) base that is linked covalently to the corrin ring. Located at the b-axial position is the sixth coordinating moiety (Gruber et al, 2011; Roth et al, 1996). This ligand can vary among cobalamin derivatives and forms a rare organometallic, covalent bond, which offers unique catalytic properties to enzymes that use Cbl as a co-factor. The two most common biological active variants have a methyl or 5’-deoxyadenosyl group at this position, resulting in methyl- and adenosyl-cobalamin (Met-Cbl and Ado-Cbl, respectively). In the industrially produced variant, a cyano-group (CN-Cbl) binds at the b-axial position, and a hydroxy group (OH-Cbl) is present in the degradation product (Gruber et al, 2011)

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