Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.
Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.
Shimoi
H
H
National Research Institute of Brewing, Tokyo, Japan.
Iimura
Y
Y
Obata
T
T
Tadenuma
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C072663:protease I, Rarobacter faecitabidus; D008358:Mannose