Molecular cloning, sequencing, and expression of the heat-labile uracil-DNA glycosylase from a marine psychrophilic bacterium, strain BMTU3346

The gene encoding a heat-labile uracil-DNA glycosylase (UDG) from a psychro philic, gram-positive marine strain (BMTU3346) has been cloned, sequenced, and expressed in Escherichia roll. The UDG is a coldactive enzyme with an a pparent temperature optimum of 35 degrees C and a half-life of 2min at 40 d egrees C. The amino acid sequence shows an identity of 39.1%-46.2% to UDGs from mesophilic bacteria. The primary structure was examined for features t hat could be related to the thermolability of the enzyme. The amino acid se quence of the heat-labile UDG shows 22 differences with respect to the cons ensus sequence derived from bacterial UDGs. Features previously recognized in cold-active enzymes such as extended surface loops or a decrease in the number of arginine residues or proline residues in loops were not observed. Because dominant features that could be related to the thermolability of t he UDG from BMTU3346 cannot be identified, more subtle modifications of the conformation seem to be responsible for its thermolability.