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Co‐ and/or post‐translational modifications are critical for TCH4 XET activity
Author(s) -
Campbell Paul,
Braam Janet
Publication year - 1998
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1998.00239.x
Subject(s) - biochemistry , xyloglucan , biology , arabidopsis thaliana , arabidopsis , mutant , enzyme , chemistry , gene
SummaryTCH4encodes a xyloglucan endotransglycosylase (XET) ofArabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co‐ and post‐translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N‐terminus are directed to the secretory pathway. Thus, as predicted, the N‐terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS‐PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylatedin vivo; glycosidases that remove N‐linked glycosylation eliminated 98% of the XET activity. Thus, co‐ and/or post‐translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site‐specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy toBacillusβ‐glucanases, this region may be the active site of XET enzymes.