Functional and Structural Characterization of an ECF-type ABC Transporter for Vitamin B12

Elife. 2018 May 29;7:e35828. doi: 10.7554/eLife.35828.

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.

Keywords: E. coli; biochemistry; chemical biology; membrane transport; molecular biophysics; structural biology; transport mechanism; vitamin B12; vitamin trasnport.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry*
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Apoproteins / chemistry*
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Biological Transport
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Folic Acid / chemistry*
  • Folic Acid / metabolism
  • Gene Expression
  • Genetic Complementation Test
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Kinetics
  • Lactobacillus delbrueckii / chemistry*
  • Lactobacillus delbrueckii / metabolism
  • Models, Molecular
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structural Homology, Protein
  • Substrate Specificity
  • Vitamin B 12 / chemistry*
  • Vitamin B 12 / metabolism

Substances

  • ATP-Binding Cassette Transporters
  • Apoproteins
  • Bacterial Proteins
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Folic Acid
  • Vitamin B 12