Characterisation of a binding-protein-dependent, active transport system for short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus.
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.
Mills
J
J
Department of Biochemistry, University of Leicester, England.
Wyborn
N R
NR
Greenwood
J A
JA
Williams
S G
SG
Jones
C W
CW
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D015964:Gene Expression Regulation, Bacterial; D016946:Gram-Negative Aerobic Rods and Cocci / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D018272:Porins / Q000235:genetics* / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014508:Urea / Q000378:metabolism*
D000577:Amides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D033902:Periplasmic Binding Proteins; D018272:Porins; C110884:fmdD protein, Methylophilus methylotrophus; C110883:fmdE protein, Methylophilus methylotrophus; C110919:fmdF protein, Methylophilus; C070135:AmiC protein, Pseudomonas aeruginosa; D014508:Urea