A periplasmic, pyridoxal-5'-phosphate-dependent amino acid racemase in Pseudomonas taetrolens.
The pyridoxal-5'-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of D: -arginine and D: -lysine by P. taetrolens.
Matsui
Daisuke
D
Department of Life Science and Biotechnology, Kansai University, Yamate-Cho, Suita, Osaka-Fu, Japan.
Oikawa
Tadao
T
Arakawa
Noriaki
N
Osumi
Shintaro
S
Lausberg
Frank
F
Stäbler
Norma
N
Freudl
Roland
R
Eggeling
Lothar
L
D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009952:Ornithine / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011549:Pseudomonas / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D001120:Arginine; D009952:Ornithine; D000591:Amino Acid Isomerases; D008239:Lysine