Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.
A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.
Binnie
C
C
Cangene Corporation, Mississauga, Canada.
Butler
M J
MJ
Aphale
J S
JS
Bourgault
R
R
DiZonno
M A
MA
Krygsman
P
P
Liao
L
L
Walczyk
E
E
Malek
L T
LT
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology; D002876:Chromosomes, Bacterial / Q000235:genetics; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D015698:Genomic Library; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008074:Lipoproteins; D009842:Oligopeptides; D010450:Endopeptidases; C096435:SlpD protein, Streptomyces lividans; C096436:SlpE protein, Streptomyces lividans; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases