Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.
To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.
Mayer
R
R
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Ross
P
P
Weinhouse
H
H
Amikam
D
D
Volman
G
G
Ohana
P
P
Calhoon
R D
RD
Wong
H C
HC
Emerick
A W
AW
Benziman
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D001419:Bacteria / Q000201:enzymology; D015153:Blotting, Western; D003429:Cross Reactions; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005964:Glucosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D010944:Plants / Q000378:metabolism*; D013379:Substrate Specificity
D000345:Affinity Labels; D029681:Arabidopsis Proteins; D010455:Peptides; C062025:bis(3',5')-cyclic diguanylic acid; D005964:Glucosyltransferases; C457769:PRC1 protein, Arabidopsis; C028634:cellulose synthase (UDP-forming); D006152:Cyclic GMP