Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi.
Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so-called Out general secretion pathway and, presumably, involves recognition of EGZ-carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N- and C-proximal sides, separated by a Ser/Thr-rich region, which are responsible for catalysis and cellulose-binding, respectively. The existence of a secretion region ('targeting signal') was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose-binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr-rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose-binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi.(ABSTRACT TRUNCATED AT 250 WORDS)
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LCB-CNRS, 31 Chemin Joseph Aiguier, Marseille, France.
Chippaux
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M
Barras
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D002482:Cellulose; C080692:endoglucanase Z; D002480:Cellulase