SEQUENCING AND EXPRESSION OF THE GENE ENCODING A COLD-ACTIVE CITRATE SYNTHASE FROM AN ANTARCTIC BACTERIUM, STRAIN DS2-3R

The gene encoding citrate synthase from a novel bacterial isolate (DS2 -3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, p urified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degr ees C, and show significant activity at 10 degrees C and below. Compar ison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The a mino acid sequence of the DS2-3R enzyme shows several features previou sly recognised in other cold-active enzymes, including an extended sur face loop, an increase in the occurrence of charged residues and a dec rease in the number of proline residues in loops. Other changes observ ed in some psychrophilic enzymes, such as a decrease in isoleucine con tent and in arginine/(arginine + lysine) content, were not seen in thi s case.