Abstract
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.
Highlights
Rarobacter faecitabidusprotease I (RPI)is a serine a thiol reagentor a protease [8,11,12],whereas the proteases protease exhibiting lytic activity toward living yeast have been suspected to play a crucial role in the initial step cells
Lytic activity, the protease activity was not Rarobacter protease I (RPI),’ themajor yeast-lytic protease affected. These findings suggest that the COOH-ter- of R. faecitabidus, has been characterized previously [18].RPI minaldomainactuallyparticipates in themannose- is a serineproteasewith a molecular weight of 35,000, is binding activity and is required for yeast-lytic activity. elastase-like in its substratespecificity, and is a trypsin-type serine protease based on the homology of its NH2-terminal sequence
The mutant RPI which was characterized was partially purified from the culturebroth of E. coli with themutant plasmid using the method described under “Purification of RPI expressed in E. coli” except for gel filtration
Summary
Its nucleotide sequence revealed thatit Rarobacter faecitabidus [15] is a yeast-lytic bacterium isocontains an open reading frame encoding62a6-amino lated from a waste water treatment system. Lytic activity, the protease activity was not Rarobacter protease I (RPI),’ themajor yeast-lytic protease affected These findings suggest that the COOH-ter- of R. faecitabidus, has been characterized previously [18].RPI minaldomainactuallyparticipates in themannose- is a serineproteasewith a molecular weight of 35,000, is binding activity and is required for yeast-lytic activity. Elastase-like in its substratespecificity, and is a trypsin-type serine protease based on the homology of its NH2-terminal sequence Because both the protease and yeast-lytic activity.
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