Purification and Characterization of Cellulose Synthase Activity

Cellulose synthase is the enzyme that performs cellulose biosynthesis. Purification of this enzyme is a major objective for understanding its properties and in determining its structure and mode of regulation. Cellulose synthase is a membrane protein and like most membrane proteins its purification has eluded investigators interested in isolating it. However, significant progress has been made in purifying the cellulose synthase activity from A. xylinum using the product entrapment technique utilized for purification of the chitin synthase activity in yeast (Lin and Brown, 1989). In A. xylinum, using a combination of detergent solubilization and product entrapment methods, two major polypeptide bands were identified in the purified fraction. One of these polypeptides was shown to selectively bind UDP-glucose, and this polypeptide was identified as the cellulose synthase catalytic subunit (Lin et al., 1990). The other polypeptide was shown to bind the activator c-di-GMP (Mayer et al., 1991). Sequence information obtained from these polypeptides was useful in identifying the corresponding genes from A. xylinum (Saxena et al., 1990, 1991). However, similar progress has not been made with purifying the cellulose synthase activity in plants. Laosinchai (2002) used immunoaffinity techniques to purify cellulose synthase activity from mung bean fractions that synthesized cellulose microfibrils in vitro (Laosinchai, 2002). Unfortunately, sufficient amounts of the protein could not be isolated for further characterization of this activity. The cellulose synthase activity purified from A. xylinum utilizes UDP-glucose as the substrate and is activated by c-di-GMP. The cellulose synthase activity in plants is also shown to use UDP-glucose as the substrate, but it is not activated by c-di-GMP. Instead, the plant activity is influenced positively in the presence of cellobiose (Li and Brown, 1993). Although no requirement for a primer has been observed for cellulose synthesis in vitro using bacterial or plant extracts, a proposal for the requirement of a sterol-glucoside primer has been made for cellulose synthesis in plants (Peng et al., 2002). This proposal is based on the observation that cotton fiber membranes synthesized sitosterol-cellodextrins (SCDs) from sitosterol-β-glucoside (SG) and UDP-glucose under conditions that favor cellulose synthesis (Peng et al., 2002).As a result, this model invokes a number of other components besides cellulose synthase and UDP-glucose, in a multistep reaction scheme, as opposed to the single-step polymerization reaction that requires only cellulose synthase and UDP-glucose. Since most of the experiments demonstrating in vitro cellulose synthesis do not suggest the requirement for a primer and no new evidence has been provided in support of the multistep reaction scheme, the current view is that polymerization of glucose residues from UDP-glucose occurs in a single-step reaction catalyzed by the cellulose synthase.

Interestingly, many of the features of cellulose synthases from different organisms are predicted from the derived amino sequences following identification of the genes for cellulose synthases in these organisms.