COMPOSITIONS AND METHODS FOR REGULATING BACTERIAL CELLULOSE SYNTHASE AND FOR SYNTHESIZING CELLULOSE IN VITRO

摘要

Disclosed herein is in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Further disclosed is that BcsB is essential for catalysis by BcsA. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity of native proteins depends on the presence of cyclic-di-GMP, but is independent of lipid-linked reactants. Further disclosed is strict substrate specificity of cellulose synthase for UDP-glucose. Truncation analysis of BcsB localized the region required for activity of BcsA within its C-terminal membrane-associated domain. Further disclosed are crystal structures of the cyclic-di-GMP-activated BcsA-B complex revealing that cyclic-di-GMP releases an auto-inhibited state of the enzyme by breaking a salt bridge which otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Unexpectedly, disrupting the salt bridge by mutagenesis generates a constitutively and fully active cellulose synthase.