Characterisation of a binding-protein-dependent, active transport system for short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus

Eur J Biochem. 1998 Jan 15;251(1-2):45-53. doi: 10.1046/j.1432-1327.1998.2510045.x.


Three genes (fmdCAB) encoding an outer-membrane porin for short-chain amides and urea, formamidase, and a putative regulatory protein in Methylophilus methylotrophus have previously been cloned and characterised. Three genes have now been identified downstream of fmdB, viz fmdD encoding a hydrophilic protein containing an N-terminal signal sequence, and fmdEF encoding hydrophobic transmembrane proteins. The derived amino acid sequence of mature FmdD (predicted molecular mass 41,870 Da) was similar to the cytoplasmic, amide-binding protein (AmiC) from Pseudomonas aeruginosa and to several periplasmic, solute-binding proteins from other bacteria. Mature FmdD was purified and shown to be a monomer (40-45 kDa) with the predicted N-terminal amino acid sequence (ADYPTA-). Equilibrium dialysis showed that the purified protein bound short-chain amides and urea with high affinity (Kd 7.2 microM for [14C]urea). SDS/PAGE and western blotting using antiserum to mature FmdD showed it was induced by short-chain amides and urea, and repressed by excess ammonia. The derived amino acid sequences of FmdE (32,822 Da) and FmdF (incomplete; >25,435 Da) were similar to the transmembrane proteins BraD/LivH and BraE/LivM, respectively, in various leucine/isoleucine/valine transport systems. Uptake of [14C]urea by washed cells was inhibited by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone and unlabelled formamide. It is concluded that FmdDEF comprise part of a high-affinity, binding-protein-dependent active-transport system for short-chain amides and urea in M. methylotrophus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides / metabolism*
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Biological Transport
  • Gene Expression Regulation, Bacterial
  • Gram-Negative Aerobic Rods and Cocci / chemistry*
  • Gram-Negative Aerobic Rods and Cocci / genetics
  • Gram-Negative Aerobic Rods and Cocci / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Periplasmic Binding Proteins*
  • Porins / genetics*
  • Porins / metabolism*
  • Sequence Analysis
  • Sequence Homology, Amino Acid
  • Urea / metabolism*


  • Amides
  • Bacterial Proteins
  • Membrane Proteins
  • Periplasmic Binding Proteins
  • Porins
  • fmdD protein, Methylophilus methylotrophus
  • fmdE protein, Methylophilus methylotrophus
  • fmdF protein, Methylophilus
  • AmiC protein, Pseudomonas aeruginosa
  • Urea

Associated data

  • GENBANK/Y14964