Impact of amino acid changes in the signal peptide on the secretion of the Tat-dependent xylanase C from Streptomyces lividans.
Xylanase C (XlnC) is a cofactorless protein secreted through the twin arginine translocation (Tat)-dependent secretion pathway by Streptomyces lividans. Its signal peptide contains the SRRGFLG sequence, which is similar to the twin-arginine consensus motif. The 49 amino acid-long signal peptide was analyzed by random, site-directed and site-saturation mutagenesis and the effect of these mutations on XlnC secretion determined. None of the mutations abolished XlnC production and the decreased yields were attributed to the low processing rate of precursors ranging from 2 to 5 h instead of 11 min for the wild-type precursor. Replacement of phenylalanine in the consensus motif by other amino acid residues decreased XlnC secretion by 75%, except for a tryptophan substitution which demonstrated a 50% decrease. Charge distribution in the n-domain of the signal peptide was more important than the net charge. Replacement of the signal peptidase recognition site A-H-A by either A-H-E, V-D-S or R-L-E did not affect precursor processing, indicating that the presence of the conserved residues found in the signal peptidase recognition site is not a prerequisite for the processing of Tat-substrates as it is for the processing of Sec-substrates in S. lividans.
Li
Haiming
H
INRS-Institut Armand-Frappier, Université du Québec, Ville de Laval, Québec, Canada.
Faury
Damien
D
Morosoli
Rolf
R
D000595:Amino Acid Sequence; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics*; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C408585:twin-arginine translocase complex, E coli; D043364:Endo-1,4-beta Xylanases